CN103509114B - For the monoclonal antibody for claudin -18 for the treatment of cancer - Google Patents

For the monoclonal antibody for claudin -18 for the treatment of cancer Download PDF

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Publication number
CN103509114B
CN103509114B CN201310314152.4A CN201310314152A CN103509114B CN 103509114 B CN103509114 B CN 103509114B CN 201310314152 A CN201310314152 A CN 201310314152A CN 103509114 B CN103509114 B CN 103509114B
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seq
antibody
amino acid
cell
acid sequence
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CN201310314152.4A
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CN103509114A (en
Inventor
乌尔·沙欣
厄兹莱姆·图雷奇
迪尔克·乌泽纳
斯特凡·弗里茨
克里斯托夫·乌赫雷克
贡达·勃兰登堡
哈拉尔德-格哈德·格佩特
安雅·克里斯蒂娜·施罗德
菲利普·蒂尔
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Johannes Gutenberger Mainz University represented by the president
Translationale Onkologie An Der Universitatsmedizin Der Johannes Gutenberg-Univers
Ganymed Pharmaceuticals GmbH
Astellas Pharma Inc
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Johannes Gutenberger Mainz University Represented By President
Ganymed Pharmaceuticals GmbH
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Abstract

The present invention provides antibody, the antibody can be used to treating and/or preventing the relevant disease of cell with expression CLD18 as therapeutic agent, including tumor-related illness, such as gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, oophoroma, colon cancer, liver cancer, head and neck cancer and gallbladder cancer.

Description

For the monoclonal antibody for claudin -18 for the treatment of cancer
It is on November 24th, 2006 applying date that the application, which is, application No. is " 200680043664.X ", entitled " use In the monoclonal antibody for claudin -18 for the treatment of cancer " Chinese patent application divisional application, original application is international Shen Please PCT/EP2006/011302 National Phase in China application.
Technical field
Compared with conventional medicine, the cancer therapy based on antibody has the potentiality compared with high specific and compared with low side effect spectrum. The reason for this is that antibody to normal cell and neoplastic cell accurately distinguish and following facts, i.e., its binding mode is dependent on poison The less immunology antitumor mechanism of property, such as the recruitment of complement activation and cell toxicant immunocyte.
Background technology
The target of therapy based on antibody must have specific character, and normal cell and superfluous life are distinguished this results in correct The basis of property cell.Obviously, exclusively it is confined to the target that can't detect in tumour cell and completely in the normal tissue It is highly desirable for developing efficient and safe antibody therapy.On the other hand, high-caliber overexpression can also be controlled The basis of window and low side effect is treated, such as due to gene magnification and as antibody trastuzumab (trastuzumab, herceptin) 2 type human epidermal growth factor acceptors (HER-2) of excellent target.
It is granted or just other targets of the antibody for tumor therapy of clinical development have be not based on target molecule The peculiar property being overexpressed in quantity in tumour cell.For being directed to the antibody of proteoglycans MUC-1, the peptide in target main chain It is hypo-glycosylated in tumour cell to repeat epitope, so as to be changed to the counterpart of its normal condition (counterpart).It is right In for CD20 (Rituximab (rituximab)), CD52 (Campath-1H) and CD22 (epratuzumabs (epratuzumab)) antibody, the expression that antibody target is marked in tumour cell and normal lymphocytes are suitable.This feelings Under condition, antibody can tolerate the removal of normal cell, because to have restored normal lymph thin for the stem cell of target feminine gender Born of the same parents compose.Other examples of antibody target otherness accessibility are carcinomebryonic antigen (carcinoembryonal antigen, CEA) With carbonic anhydrase IX (carboanhydrase IX, CA9).Both antigens are expressed in the normal epithelial of colon and kidney respectively. However, radiolabeled imaging antibody certain good discrimination tumour and normal structure, and cytotoxic antibody can be with Tolerate well.This is likely due to CA9 and the CEA restricted expression in normal epithelial tissues can not approach in IgG antibody Chamber side.Epithelial cell adhesion molecule (epithelial cell adhesion molecule, Ep-CAM) this antigen also belongs to In this classification.As the homocellular adhesion molecule of epithelial cell, it is positioned in space between cells.Ironically, to the greatest extent The anti-Ep-CAM antibody toxicity of pipe high-affinity is high, but the antibody of intermediate affinity is well to tolerate.This has prompted Ep- Accessibility of the CAM targets on normal cell, but also indicate that antibody binding kinetics provide possibility for treatment.
A kind of possibility is to be related to other epithelial cell specific proteins of cell/cell adherence for antibody method It is significant, because they almost can not be close to antibody in the epithelium of normal configuration, but it is exposed in tumour cell.Cause This, we analyze the protein for participating in tissue epithelial institutional framework as the applicability of therapeutic antibodies target.One Kind especially attracts we note that the protein of power is claudin 18 (claudin 18).
Claudin 18 (CLD18) molecule (Genbank registration numbers：Splice variant 1 (CLD18A1)：NP_057453、 NM016369 and splice variant 2 (CLD18A2)：NM_001002026, NP_001002026) be molecular weight it is about 27,9/ The inherent transmembrane protein of 27,72kD.Claudin is the inherent memebrane protein in the close connection of epithelium and endothelium.Close connection The net of intramembranous particles interconnection chain is organized between flanking cell.In close connection, Occludin (occludin) and claudin It is most important transmembrane protein component.Due to its strong Intercellular Adhesion characteristic, they are produced by the cell for preventing and controlling solute It transports and limits film fat and protein sideways diffusion is to maintain the primary barrier of cell polarity.Close-connected protein is formed to close Take part in keyness tissue epithelial institutional framework.It is presumed that these protein can not almost connect in good epithelium is constructed Nearly antibody, but then become to be exposed in tumour cell.
CLD18 is a kind of tetratransmembrane albumen, therefore there are four hydrophobic regions for tool.It is that we obtain statistics indicate that CLD18 exhibitions Show several tripe systems as these conformations can selectively be addressed by antibody.A kind of conformation (CLD18- conformations -1) display, all four Hydrophobic region is as conventional transmembrane region (transmembrane domain, TM), and (ring 1 is by hydrophobic region 1 for two extracellular rings of formation It surround and forms with hydrophobic region 2；Ring 2 is surround by hydrophobic region 3 and hydrophobic region 4 and formed), as to most claudin family member institutes As description.Another conformation (CLD18- conformations -2) display, as described to another tetratransmembrane family member PMP22 (Taylor etc., J.Neurosc.Res.62 like that：15-27,2000), second and third hydrophobic domains not fully across Plasma membrane, so as to which the part (ring D3) between first and the 4th transmembrane domain is extracellular.The third conformation (CLD18- structure As -3) show that there are two inner hydrophobics by surrounding tool by first and the 4th hydrophobic region as conventional transmembrane domain The big extracellular domain in area.Due to there is classical N- glycosylation sites, the topology variant CLD18 of claudin -18 in ring D3 - 2 and CLD18 of topology topologys -3 are with additional extracellular N- glycosylation sites.
The presence of two kinds of different splice variants adds the complexity in another layer of level to CLD18 molecules, both changes Body is described in mouse and people (Niimi, Mol.Cell.Biol.21：7380-90,2001).Splice variant CLD18A1 and CLD18A2 has differences before the N-terminal including first TM and ring 1 in 21 amino acid, and the primary protein sequences phase of C-terminal Together.
CLD18A1 selective expressions in normal pneumogastric epithelium, and CLD18A2 is only expressed in gastric cells (Niimi, Mol.Cell.Biol.21：7380-90,2001).Most of all, CLD18A2 is confined to differentiated Weishang skin In short-lived cell, but it is not present in stomach stem cell area.We show that two kinds of variants are at any other using sensitive RT-PCR It can't detect completely, but the strong expression in several cancer types in normal human organs, including stomach, oesophagus, pancreas and lung Tumour and human cancer cell line.Expression is mainly in the adenocarcinoma subtypes of these indications.
The molecular weight of the protein has differences in certain cancers and neighbouring normal structure.It is observed in health tissues To higher molecular weight protein can by using deglycosylation compound PNGase F handle Tissue lysates and be transformed into With identical molecular weight observed in cancer.This prompting is compared with its normal structure counterpart, CLD18 N- glycosyls in cancer Change less.This architectural difference is likely to generate the epitope changed.Classical N- glycoylation motifs are in Molecular Ring D3 structural domains 116 amino acids in.
The term " CLD18 " and " CLD18 variants " of the present invention should include (i) CLD18 splice variants；(ii) CLD18 N sugar Base variant；(iii) CLD18 conformational variants thereofs；(iv) positioned at intercellular closely connect in free CLD18 be connected with homotype/abnormal shape The non-cancer cell related variants of the cancer related variants and CLD18 of variant and (v) CLD18.
The characterization of molecules and functional character of CLD18 so that the molecule is very significant to the cancer therapy based on antibody.It is special It is not that there is no CLD18 in (i) most toxicity correlation normal structures；(ii) expression of CLD18 A2 variants is confined to conduct In the dispersible cell colony of differentiated gastric cells, these cells can be supplemented by the stomach stem cell of target feminine gender；(iii) just The potential glycosylated clue of otherness between normal and neoplastic cell；(iv) tripe systems are as the presence of topology situation.In addition, CLD18 may further contribute to good therapeutic window as the effect of tight junction protein.Due to tumor cells expression claudin, But often the close of classics is formed by the way that the homotype of claudin and abnormal shape are associated unlike as visible in normal epithelial tissues Connection, therefore tumour cell has the considerable free claudin library for being suitable for the combination of extracellular antibody and immunotherapy.It is possible that The combination epitope of claudin is shielded in close connection in healthy epithelium, makes these antibody that can not approach.
It is an object of the present invention to provide the diseases (such as tumor disease) for wherein having CLD18 expression available for treatment Antibody.Antibody described herein can also be used to diagnose these diseases.
Invention content
Present invention generally provides antibody, the antibody can be used as therapeutic agent for treating and/or preventing and expression CLD18 The relevant disease of cell, including tumor-related illness, such as gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, oophoroma, colon cancer, liver Cancer, head and neck cancer and gallbladder cancer.
One aspect of the present invention relates to the antibody of cell for combining CLD18 and mediating killing expression CLD18.It is preferred that Ground, the antibody are combined with CLD18A1 and CLD18A2, it is highly preferred that the antibody is combined with CLD18A2 but do not tied with CLD18A1 It closes.Preferably, antibody of the invention is combined with the ring 1 or ring 2 of CLD- conformations -1 and has specificity to it.In another preferred reality Apply in scheme, antibody of the invention combined with the ring D3 of CLD- conformations -2 and to its have specificity, particularly with 116 in ring D3 It is combined at the N glycosylation sites of position or around it.In another embodiment, antibody of the invention is to 116 potential N in ring D3 Glycosylated form, which does not occur, for glycosylation site has specificity.
The killing of antibody on cell of the present invention preferably is combined to lure by the antibody with the CLD18 expressed by the cell It leads, more preferably is combined to induce with the CLD18A2 expressed by the cell by the antibody.In one embodiment, this hair The combination of CLD18A1 expressed by bright antibody and the cell or not described in induced killer.
The cell of the expression CLD18 is preferably cancer cell, is especially selected from stomach, oesophagus, pancreas, lung, the ovum of oncogenicity Nest, colon, liver, neck and bladder cancer cell.
Preferably, antibody of the present invention is mediated by inducing complement dependent cellular cytotoxicity (CDC) cracking, antibody dependent Cracking, apoptosis, homotypic adhesion and/or the phagocytosis of cytotoxicity (ADCC) mediation mediate the killing to cell, preferably logical The cracking of induction CDC mediations and/or the cracking of ADCC mediations are crossed to mediate the killing to cell.
In one embodiment, antibody of the invention does not induce the cell cracking that CDC is mediated.
Preferably, the cell cracking of ADCC mediations occurs in the presence of effector cell, and the effector cell is being embodied Make in scheme selected from monocyte (monocyte), mononuclearcell (mononuclear cell), NK cells and PMN, phagocytosis With being realized by macrophage.
The antibody of the present invention can be monoclonal antibody, chimeric antibody, human antibody or humanized antibody either antibody piece Section, and may be selected from IgG1, IgG2 (preferably IgG2a and IgG2b), IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD and IgE antibody.
All aspects according to the present invention, CLD18 are preferably people CLD18, preferably people CLD18A2, and CLD18A2 is preferred With SEQ ID NO：2 amino acid sequence, CLD18A1 preferably have SEQ ID NO：8 sequence.
In an especially preferred embodiment, the day of CLD18 present on antibody and liver cell surface of the invention Right epitope combines.In another preferred embodiment, antibody of the invention has cancer cell (preferably stomach cancer cell) special Property.
In certain embodiments of the invention, CLD18 is expressed in cell surface.
The antibody of the present invention can be obtained by method comprising the following steps：With with selected from SEQ ID NO：2、4、6、 16th, 18,20, the protein of the amino acid sequence of 21-23 and 26-31 or polypeptide or its immunogenic fragments or the expression egg Animal is immunized in the nucleic acid or host cell of white matter or polypeptide or its immunogenic fragments.Preferably, antibody of the invention is to above-mentioned Protein, polypeptide or its immunogenic fragments have specificity.
In an especially preferred embodiment, antibody of the invention is DSM ACC2737 (182- by registration number D1106-055)、DSM ACC2738(182-D1106-056)、DSM ACC2739(182-D1106-057)、DSM ACC2740 (182-D1106-058)、DSM ACC2741(182-D1106-059)、DSM ACC2742(182-D1106-062)、DSM ACC2743(182-D1106-067)、DSM ACC2745(182-D758-035)、DSM ACC2746(182-D758-036)、 DSM ACC2747(182-D758-040)、DSM ACC2748(182-D1106-061)、DSM ACC2808(182-D1106- 279), the clone of DSM ACC2809 (182-D1106-294) or DSM ACC2810 (182-D 1106-362) generates.
In one embodiment, antibody of the invention and therapeutic agent such as toxin, radioactive isotope, drug or cell toxicant Agent is coupled.
Another aspect of the present invention relates to the hybridomas that can generate antibody of the present invention.It is preferred that the registration number of hybridoma is DSM ACC2737(182-D1106-055)、DSM ACC2738(182-D1106-056)、DSM ACC2739(182-D1106-057)、 DSM ACC2740(182-D1106-058)、DSM ACC2741(182-D1106-059)、DSM ACC2742(182-D1106- 062)、DSM ACC2743(182-D1106-067)、DSM ACC2745(182-D758-035)、DSM ACC2746(182- D758-036)、DSM ACC2747(182-D758-040)、DSM ACC2748(182-D1106-061)、DSM ACC2808 (182-D1106-279), DSM ACC2809 (182-D1106-294) or DSM ACC2810 (182-D1106-362).
The present invention antibody herein by reference in the antibody name (such as 182-D758-035) and/or by reference to The clone (such as 26D12) of the antibody is generated to name.
The invention further relates to the drugs comprising antibody of the present invention and/or its conjugate and pharmaceutical acceptable carrier with therapeutic agent Composition.
CLD18 (preferably CLD18A2) is expressed another aspect of the present invention relates to killing and/or inhibits the method for growth, Antibody of the present invention and/or its conjugate with therapeutic agent including making cell with effective amounts.CLD18 is preferably in the cell Surface expression.
Another aspect of the present invention relates to the relevant diseases of cell treated or prevented with expression CLD18 (preferably CLD18A2) The method of disease or illness, including to the antibody of the subject application present invention, its conjugate with therapeutic agent or including the present invention The pharmaceutical composition of antibody or its conjugate with therapeutic agent.Preferably, the disease or illness are tumor-related illness, are being had Gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, oophoroma, colon cancer, liver cancer, head and neck cancer and gallbladder cancer are selected from body embodiment. CLD18 is preferably in the surface expression of the cell.
Preferably, it is expressed can to distinguish different cell types (including cancer cell and non-malignant cell) to antibody of the invention CLD18 variants.In an especially preferred embodiment, antibody of the invention can be combined with CLD18A2, but not with CLD18A1 combinations or the specificity combined with CLD18A1 are less than the binding specificity with CLD18A2.
The term " with reference to " of the present invention is preferably directed to specifically bind.It is special with it that " specific binding " refers to reagent (such as antibody) The combination of specific target (such as epitope) is better than and the combination of other targets.If the dissociation constant that reagent is combined with the first target (KD) less than the dissociation constant with second of target, then the combination of itself and the first target is better than and the combination of second of target. Preferably, the dissociation constant (K of the target of reagent specific bindingD) normal less than the dissociation of the target of the reagent non-specific binding Number (KD) 1/10 hereinafter, it is preferred that 1/20 hereinafter, more preferable 1/50 hereinafter, even more preferably 1/100,1/200,1/500 or 1/ Less than 1000.
The antibody of the present invention mediates killing expression CLD18 (excellent by combining CLD18 (preferred expression is in described cell surface) Choosing expression CLD18A2) cell.In one embodiment, antibody induction complement-dependent cytotoxicity of the invention (CDC), such as the cracking that CLD18 expression cells generation at least about 20-40%CDC is mediated, what preferably from about 40-50%CDC was mediated splits Solution, the cracking of more preferably higher than 50%CDC mediations.Such antibody carries out example herein by following antibody：37H8、 38G5,38H3,39F11,61C2,26B5,26D12,28D10,163E12,175D10,45C1,125E1, ch-163E12 and ch- 175D10.As the substituted or supplemented of induction CDC, antibody of the invention can effector cell (such as monocyte (monocyte), Mononuclearcell (mononuclear cell), NK cells and PMN) in the presence of induce CLD18 expression cells antibody dependent Cytotoxicity (ADCC).Such antibody carries out example herein by following antibody：37G11、37H8、38G5、38H3、 39F11,43A11,61C2,26B5,26D12,28D10,42E12,163E12,175D10,45C1 and 125E1.The present invention's is anti- Body can have following ability：It induces the apoptosis of CLD18 expression cells, induce the homotypic adhesion of CLD18 expression cells and/or huge The phagocytosis of CLD18 expression cells is induced in the presence of phagocyte.The antibody of the present invention can have one or more above-mentioned functions Characteristic.Preferably, the cracking of the CDC mediations of antibody induction CLD18 expression cells of the invention and the cracking of ADCC mediations, it is more excellent The cracking of the ADCC mediations of choosing induction CLD18 expression cells, the cracking without inducing the cell CDC mediations.Antibody of the present invention Exemplary target cell include but are not limited to expression CLD18 (preferably CLD128A2) cancer cell, stomach, pancreas such as oncogenicity The cancer cell of gland, oesophagus and lung.In a specific preferred embodiment, the antibody-mediated cell killing of the present invention is CLD18A2 specificity, i.e., antibody-mediated killing (preferably CDC and/or the cell cracking of ADCC mediations) expression of the invention The cell of CLD18A2, but cells of the killing expression CLD18A1 without expressing CLD18A2 is not mediated.Above-mentioned antibody can be for example It is used to mediate killing tumor cell in the treatment or prevention of following cancer：Gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, oophoroma, knot Intestinal cancer, liver cancer, head and neck cancer and gallbladder cancer.
The antibody of the present invention can be according to its binding characteristic and its mediation effector to the ability of the function of CLD18 expression cells Be categorized into it is different classes of in.Antibody of the present invention can classify according to following characteristics：
● to express CLD18A1 or CLD18A2 the binding characteristic of cell and/or the effector of mediation to the cell Function (distinguishes CLD18 splice variants),
● to expression glycosylation or the binding characteristic of cell of non-glycosylated CLD18 variants and/or the effector pair of mediation The function (differentiation whether there is the glycosylated CLD18 variants of N) of the cell,
● the effector of binding characteristic and/or mediation to the cell of cancer cell or normal cell types is to the cell Function (distinguishes tumor cells expression or normal cell expression CLD18 variants),
● to the binding characteristic of CLD18 epitopes that the formation being tightly coupled is shielded,
● the ability that formed to living cells induction CLD18 aggregations and
● the ability combined with inhuman CLD18 variants especially from the CLD18 of mouse, rat, rabbit and primate.
The present invention antibody have one or more following characteristics, wherein provide antibody of the present invention (24H5,26B5, 26D12、28D10、37G11、37H8、38G5、38H3、39F11、41C6、42E12、43A11、44E10、47D12、61C2、 75B8、85A3、9E8、19B9、45C1、125E1、163E12、166E2、175D10、ch-43A11、ch-45C1、ch-125E1、 Ch-163E12, ch-166E2, ch-175D10) specific example as reference：
A) it is combined with CLD18A2 and CLD18A1 (such as 26D12,28D10,37H8,38H3,39F11,61C2 and 41C6)
B) combined with CLD18A2 but do not combined with CLD18A1 (such as 26B5,37G11,38G5,42E12 and 43A11,45C1, 125E1、163E12、166E2、175D10、ch-43A11、ch-45C1、ch-125E1、ch-163E12、ch-166E2、ch- 175D10)
C) it is combined with the CLD18 that tumour cell is naturally expressed but not natural with non-cancerous cells or tissue (such as stomach and pneumonocyte) The CLD18 of expression combines (such as 26B5,75B8,24H5,39F11,45C1,125E1,163E12,166E2,175D10)
D) killing for expression CLD18A2 cells of mediation CDC inductions, without being directed to the cell of expression CLD18A1 (such as 26D12,28D10,37H8 and 39F11,163E12, ch-125E1, ch-163E12, ch-175D10)
E) mediation ADCC induction to express CLD18 cells killing (such as 26B5,37G11,37H8,38G5,38H3, 39F11,43A11,47D12 and 61C2, ch-163E12, ch-175D10)
F) mediation ADCC induction to express CLD18 cells killing, but do not mediate CDC mediate killing (such as 37G11, 42E12 and 43A11)
G) mediation ADCC induction and CDC induction to express CLD18A2 cells killing (such as 37H8,38H3,39F11, ch-163E12、ch-175D10)。
As described herein exemplary, antibody of the invention further includes following molecule,
A) with differentiated normal gastric cell combination, but (such as 39F11) is not combined with stomach stem cell
B) it is not combined with normal gastric mucosa and other normal organs, but (such as 26B5) is exclusively combined with cancer cell
C) epitope with 116 non-glycosylated Asn comprising CLD18 is combined
D) it is combined with people and mouse CLD18 so that preclinical toxicity research can be fully carried out in mouse.
The antibody of the present invention may be from different plant species, include but are not limited to mouse, rat, rabbit, cavy and people.The present invention Antibody further include chimeric molecule, wherein the antibody constant region from a species (preferably people) and the antigen from another species Binding site combines.In addition, the antibody of the present invention includes humanized molecule, wherein the antigen binding of the antibody from non-human species It is combined with the constant region and framework region of people source in site.
The antibody of the present invention includes polyclonal antibody and monoclonal antibody, and including IgG2a (such as IgG2a, κ, λ), IgG2b (such as IgG2b, κ, λ), IgG3 (such as IgG3, κ, λ) and IgM antibody.However, the present invention also includes other antibody isotypes, including IgG1, IgA1, IgA2, secretory IgA, IgD and IgE antibody.The antibody can be complete antibody or its antigen-binding fragment, Including such as Fab, F (ab ')2, Fy, single-stranded Fy segments or bispecific antibody.In addition, the antigen-binding fragment includes Comprising with the binding structural domain domain-immunoglobulin fusion proteins of lower part：(i) knot merged with immunoglobulin hinge region polypeptide Close Domain Polypeptide (such as heavy chain variable region or light chain variable region), the heavy chain immunoglobulin CH2 that (ii) is merged with the hinge area The heavy chain immunoglobulin CH3 constant regions of constant region and (iii) and the CH2 constant domains.Such binding structural domain is immunized Immunoglobulin fusion albumen has further disclosure in US2003/0118592 and US 2003/0133939.
The antibody of the present invention is preferably with about 1-100nM or smaller Dissociation equilibrium constants (KD) dissociated with CLD18.It is preferred that Ground, antibody of the invention with associated cell surface antigen cross reaction, therefore do not inhibit its function.
In some preferred embodiments, antibody of the invention can be characterized by one or more following characteristics：
A) to the specificity of CLD18, particularly to the specificity of CLD18A2；
B) to the CLD18 particularly binding affinities of CLD18A2 it is about 100nM or smaller, preferably from about 5-10nM or smaller, More preferably from about 1-3nM or smaller,
C) mediation is to the ability of the high-level CDC of CD55/59 feminine genders or CD55/59 positive cells；
D) inhibit the ability of the cell growth of expression CLD18；
E) ability of the Apoptosis of induced expression CLD18；
F) ability to the cell induction homotypic adhesion for expressing CLD18；
G) ability in the presence of effector cell to the cell induction ADCC for expressing CLD18；
H) extend the ability of subject's survival of the tumour with expression CLD18；
I) ability of the cell of expression CLD18 is removed；
J) remove the cell of expression low-level CLD18 ability and/or
K) in the ability of liver cell surface aggregation CLD18.
The anti-CLD18 antibody of the present invention can be derived, connected or be co-expressed to obtain other binding specificities.One In a specific embodiment, the present invention provides bispecific or multispecific molecule, it includes at least one to the first of CLD18 Second binding specificity of binding specificity (such as anti-CLD18 antibody or its analogue body) and pairing effect cell, such as to Fc receptors The binding specificity of (such as Fc- γ receptors, such as Fc- γ RI or other Fc receptors) or T cell receptor (such as CD3).
Therefore, the present invention include the bispecific combined with CLD18 and Fc receptors or T cell receptor (such as CD3) and Multispecific molecule.The example of Fc receptors is IgG receptors, Fc- γ receptors (Fc- γ R) such as Fc- γ RI (CD64), Fc- γ RII (CD32) and Fc- γ RIII (CD16).Also other Fc receptors can be targeted, such as IgA receptors (such as Fc α RI).The Fc receptors are preferred On the surface of effector cell such as monocyte, macrophage or activation mononuclearcell.In a preferred embodiment In, the bispecific and multispecific molecule are different from immunoglobulin Fc (such as IgG or IgA) binding site in Fc receptors Site at combined with this receptor.Therefore, the combination of the bispecific and multispecific molecule is not by the immune of physiological level Globulin is closed.
In another embodiment, anti-CLD18 antibody of the invention and other functional moleculars such as other peptides and protein are (such as Fab ' segments) derived, connected or co-expressed.For example, the antibody of the present invention can be with other one or more molecular entities Functionality connection (such as passing through chemical coupling, genetic fusion, non-covalent linking etc.), the molecular entity such as other antibody (as generating bispecific or multi-specificity antibody), toxin, cell ligand or antigen (as generating immunoconjugates, Such as immunotoxin).The present invention antibody can be connect with other treatment part, as radioactive isotope, small molecule anticancer drug, Recombinant cytokine or chemotactic factor (CF).Therefore, the present invention include Multiple Antibodies conjugate, bispecific or multispecific molecule and Fusion protein, they are combined with CLD18 expression cells, and available for other are molecular targeted to these cells.
In another aspect, the present invention provides composition, such as drug and diagnosis composition/kit, it includes with one kind Antibody of the present invention or antibody combination of the present invention pharmaceutical acceptable carrier formulated together.In a specific embodiment, the combination Object includes antibody combination, and the antibody combines with different epitope or with different functional character, for example, induction CDC with/ Or ADCC and apoptosis-induced.In this embodiment of the present invention, antibody can be used in combination, for example, as comprising two kinds or The pharmaceutical composition of more kinds of anti-CLD18 monoclonal antibodies uses.For example, it can will resist with different but complementation active CLD18 antibody combinations are in single therapy, to realize required curative effect.In a preferred embodiment, the composition includes Mediate the combination of the anti-CLD18 antibody and other apoptosis-induced anti-CLD18 antibody of CDC.In another embodiment, the combination Object is included in the anti-CLD18 antibody of mediated high-efficient killing target cell and inhibition CLD18 expression cell growths in the presence of effector cell Other anti-CLD18 antibody combination.
The invention also includes the anti-CLD18 antibody for simultaneously or sequentially applying two or more present invention, wherein at least one The antibody is inosculating antibody CLD18 antibody, and other at least one antibody are behaved anti-CLD18 antibody, the antibody combination CLD18 In identical or different epitope.Preferably, the chimeric CLD18 antibody of the present invention is applied first, is resisted using the people of the present invention thereafter CLD18 antibody, wherein the anti-preferred chronic administration of CLD18 antibody of the people, i.e., as maintenance therapy.
Antibody, immunoconjugates, bispecific and the multispecific molecule and composition of the present invention can be used for inhibiting table Up to the cell growth of CLD18 (particularly CLD18A2) and/or the cell of selective killing expression CLD18 (particularly CLD18A2) A variety of methods in, the method is by making the antibody, immunoconjugates, bispecific of the cell with effective amounts/mostly special Opposite molecule or composition are so as to inhibit the growth of cell and/or kill cell to realize.In one embodiment, the side Method include killing expression CLD18 cell, optionally killed in the presence of effector cell, for example, by CDC, apoptosis, ADCC, phagocytosis or the combination for passing through two or more these mechanism.It can inhibit or kill using antibody of the present invention CLD18 expression cells include cancer cell, as the stomach of oncogenicity, pancreas, oesophagus, lung, ovary, colon, liver, neck and gall-bladder are thin Born of the same parents.
Therefore, antibody of the invention can be used for treating and/or preventing a variety of diseases for being related to CLD18 expression cells, this is logical Patient's administration of antibodies to suffering from these diseases is crossed to realize.The Exemplary diseases that (as improve) or prevention can be treated are included but not It is only limitted to tumorigenic disease.The example of tumorigenic disease that can be treated and/or prevent includes gastric cancer, cancer of pancreas, the cancer of the esophagus, lung Cancer, oophoroma, colorectal cancer, liver cancer, head and neck cancer and gallbladder cancer.
In one embodiment of the invention, the subject of administration of antibodies is also in addition with chemotherapeutant, radiation Or adjusting (as enhanced or inhibiting) Fc receptors (such as Fc- γ receptors such as cell factor) expression or active medicament are treated.With Include granulocyte colony stimulating factor (G-CSF), granulocytes-macrophages collection in the typical cells factor applied during treatment G-CSF (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factor (TNF).Typical treatment agent includes antitumor Agent such as adriamycin, cis-platinum, Docetaxel, 5 FU 5 fluorouracil, methotrexate, gemcitabine and cyclophosphamide.
In another embodiment, the present invention relates to employment CLD18 or its peptide fragment to be preferably CLD18A2 or its peptide fragment Immunizing non-human animals such as mouse is to obtain the immunization strategy of antibody.It is selected from SEQ ID NO for immune preferred peptide：2、4、6、 16th, 18, the peptide of 20-23 and 26-31.Therefore, in some preferred embodiments, antibody of the invention be by using selected from SEQ ID NO：2nd, 4,6,16,18, the peptide of 20-23 and 26-31 carries out antibody that is immune and obtaining.It similarly, can be inhuman The antibody of diagnosis CLD18 is generated in transgenic animals such as transgenic mice.The non-human transgenic animal can be in genome Include all or part of heavy chain transgene of antibody of coding and the mouse of chain transgene.
The purified or enrichment of CLD18 antigens and/or nucleic acid and/or the cell of expression CLD18 or its peptide fragment can be used Preparation be immunized wild type and non-human transgenic animal.Preferably, the non-human animal can be by carrying out V-D-J recombinations It is converted with isotype and generates a variety of human monoclonal antibodies isotypes (such as IgG, IgA and/or IgM) to CLD18.Isotype turns Changing can be occurred by such as classical or non-classical isotype conversion.
Therefore, in another aspect, the present invention provides the B cell of the separation from above-mentioned non-human animal.Then can pass through It is merged with immortalized cells and immortalizes the B cell detached, to provide the source (such as hybridoma) of antibody of the present invention.This The hybridoma (hybridoma for generating antibody of the present invention) of sample is also included in the scope of the present invention.
Example as shown here, antibody of the invention can be derived directly from expressing the hybridoma of the antibody or can clone And it is recombinantly expressed in host cell (such as Chinese hamster ovary celI or lymphocyte).Other examples of host cell be microorganism, such as large intestine Bacillus and fungi, such as yeast.As replacement, they can recombinate generation in non-human transgenic animal or plant.
For generate the preferred hybridoma of antibody of the present invention be DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany；New address：Inhoffenstr.7B, 31824 Braunschweig, Germany) sequencing or The hybridoma of preservation, name and registration number are as follows：
A.182-D1106-055, registration number DSM ACC2737 are preserved on October 19th, 2005
B.182-D1106-056, registration number DSM ACC2738 are preserved on October 19th, 2005
C.182-D1106-057, registration number DSM ACC2739 are preserved on October 19th, 2005
D.182-D1106-058, registration number DSM ACC2740 are preserved on October 19th, 2005
E.182-D1106-059, registration number DSM ACC2741 are preserved on October 19th, 2005
F.182-D1106-062, registration number DSM ACC2742 are preserved on October 19th, 2005
G.182-D 1106-067, registration number DSM ACC2743 are preserved on October 19th, 2005
H.182-D758-035, registration number DSM ACC2745 are preserved on November 17th, 2005
I.182-D758-036, registration number DSM ACC2746 are preserved on November 17th, 2005
J.182-D758-040, registration number DSM ACC2747 are preserved on November 17th, 2005
K.182-D1106-061, registration number DSM ACC2748 are preserved on November 17th, 2005
L.182-D1106-279, registration number DSM ACC2808 are preserved on October 26th, 2006
M.182-D1106-294, registration number DSM ACC2809 are preserved on October 26th, 2006
N.182-D1106-362, registration number DSM ACC2810 are preserved on October 26th, 2006.
Currently preferred antibody is the antibody that above-mentioned hybridoma is generated and be available from by above-mentioned hybridoma --- i.e. pair It is 37G11 in the situation of 182-D1106-055, is 37H8 in the case of 182-D1106-056, for 182-D1106-057 Situation for 38G5, be 38H3 in the case of 182-D1106-058, be 39F11 in the case of 182-D1106-059, it is right It is 43A11 in the situation of 182-D1106-062, is 61C2 in the case of 182-D1106-067, for 182-D758-035's Situation is 26B5, is 26D12 in the case of 182-D758-036, is 28D10 in the case of 182-D758-040, for The situation of 182-D1106-061 is 42E12, is 125E1 in the case of 182-D1106-279, for 182-D1106-294's Situation is 163E12, is 175D10 in the case of 182-D1106-362 --- and its chimeric and humanization form.
In some preferred embodiments, antibody of the invention (the particularly antibody of chimeric versions thereof) is as follows including containing The antibody of heavy chain constant region (CH), the heavy chain constant region includes the amino acid sequence from people's heavy chain constant region, such as SEQ ID NO：Amino acid sequence shown in 46 or 150 or its segment.In other preferred embodiments, antibody of the invention is (particularly The antibody of chimeric versions thereof) including containing the antibody of following constant region of light chain (CL), the constant region of light chain is included from people's light chain The amino acid sequence of constant region, such as SEQ ID NO：Amino acid sequence shown in 41 or 148 or its segment.It is specific preferred at one In embodiment, antibody of the invention (the particularly antibody of chimeric versions thereof) is including containing following heavy chain constant region (CH) and containing The antibody of following constant region of light chain (CL), the heavy chain constant region include the amino acid sequence from people's heavy chain constant region, such as SEQ ID NO：Amino acid sequence shown in 46 or 150 or its segment, the constant region of light chain include the amino from people's constant region of light chain Acid sequence, such as SEQ ID NO：Amino acid sequence shown in 41 or 148 or its segment.
Include SEQ ID NO：The CH of amino acid sequence shown in 46 can be by including SEQ ID NO：Nucleic acid sequence shown in 45 Nucleic acid encode.Include SEQ ID NO：The CH of amino acid sequence shown in 150 can be by including SEQ ID NO：Nucleic acid sequence shown in 149 Nucleic acid encode.Include SEQ ID NO：The CL of amino acid sequence shown in 41 can be by including SEQ ID NO：Nucleic acid sequence shown in 40 Nucleic acid encode.Include SEQ ID NO：The CL of amino acid sequence shown in 148 can be by including SEQ ID NO：Nucleic acid sequence shown in 147 The nucleic acid encode of row.
In certain preferred aspects, the antibody of chimeric versions thereof includes such antibody, it includes heavy chain and/or gently Chain, the heavy chain contain selected from SEQ ID NO：115th, 116,117,118,119,120 and its segment amino acid sequence, it is described Light chain contains selected from SEQ ID NO：121st, 122,123,124,125,126,127,128,129 and its segment amino acid sequence Row.
In certain preferred aspects, the antibody of chimeric versions thereof include such antibody, it includes selected from it is following can The combination of the heavy chain and light chain of energy property (i) to (ix)：
(i) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 115 or its segment and include SEQ ID NO：122 The light chain of shown amino acid sequence or its segment,
(ii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 116 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 121 or its segment,
(iii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 117 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 123 or its segment,
(iv) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 119 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 126 or its segment,
(v) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 118 or its segment and include SEQ ID NO：125 The light chain of shown amino acid sequence or its segment,
(vi) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 124 or its segment,
(vii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 127 or its segment,
(viii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 128 or its segment and
(ix) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 or its segment and include SEQ ID NO： The light chain of amino acid sequence shown in 129 or its segment.
" segment " or " segment of amino acid sequence " being used above is related to a part for antibody sequence, that is, represents N and/C The sequence of the antibody sequence shortened is held, retains knot of the antibody to CLD18 in the antibody sequence in replacing antibody It closes, and preferably retains antibody function as described herein, such as the cracking of CDC mediations or the cracking of ADCC mediations.It is preferred that Ground, the segment of amino acid sequence contain from the amino acid sequence at least 80%, preferably at least 90%, 95%, 96%, 97%th, 98% or 99% amino acid residue.Selected from SEQ ID NO：115,116,117,118,119,120,121,122, The segment of 123,124,125,126,127,128 and 129 amino acid sequence be preferably directed to wherein to eliminate N-terminal 17,18,19, 20th, the sequence of 21,22 or 23 amino acid.The segment of amino acid sequence described herein can be respectively by encoding the amino acid The fragment coding of the nucleic acid sequence of sequence.
Include SEQ ID NO：The heavy chain of amino acid sequence shown in 115 can be by including SEQ ID NO：Nucleic acid sequence shown in 100 The nucleic acid encode of row.Include SEQ ID NO：The heavy chain of amino acid sequence shown in 116 can be by including SEQ ID NO：Core shown in 101 The nucleic acid encode of acid sequence.Include SEQ ID NO：The heavy chain of amino acid sequence shown in 117 can be by including SEQ ID NO：102 institutes Show the nucleic acid encode of nucleic acid sequence.Include SEQ ID NO：The heavy chain of amino acid sequence shown in 119 can be by including SEQ ID NO： The nucleic acid encode of nucleic acid sequence shown in 104.Include SEQ ID NO：The heavy chain of amino acid sequence shown in 118 can be by including SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 103.Include SEQ ID NO：The heavy chain of amino acid sequence shown in 120 can be by including SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 105.
Include SEQ ID NO：The light chain of amino acid sequence shown in 122 can be by including SEQ ID NO：Nucleic acid sequence shown in 107 The nucleic acid encode of row.Include SEQ ID NO：The light chain of amino acid sequence shown in 121 can be by including SEQ ID NO：Core shown in 106 The nucleic acid encode of acid sequence.Include SEQ ID NO：The light chain of amino acid sequence shown in 123 can be by including SEQ ID NO：108 institutes Show the nucleic acid encode of nucleic acid sequence.Include SEQ ID NO：The light chain of amino acid sequence shown in 126 can be by including SEQ ID NO： The nucleic acid encode of nucleic acid sequence shown in 111.Include SEQ ID NO：The light chain of amino acid sequence shown in 125 can be by including SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 110.Include SEQ ID NO：The light chain of amino acid sequence shown in 124 can be by including SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 109.Include SEQ ID NO：The light chain of amino acid sequence shown in 127 can by comprising SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 112.Include SEQ ID NO：The light chain of amino acid sequence shown in 128 can be by Include SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 113.Include SEQ ID NO：The light chain of amino acid sequence shown in 129 It can be by including SEQ ID NO：The nucleic acid encode of nucleic acid sequence shown in 114.
In a preferred embodiment, antibody of the invention includes heavy chain variable region (VH), contains selected from SEQ ID NO：132,133,134,135,136,137 and its amino acid sequence of segment.
In a preferred embodiment, antibody of the invention includes light chain variable region (VL), contains selected from SEQ ID NO：138,139,140,141,142,143,144,145,146 and its amino acid sequence of segment.
In certain preferred aspects, antibody of the invention includes such antibody, and it includes selected from following possibility The combination of the heavy chain variable region (VH) and light chain variable region (VL) of property (i) to (ix)：
(i) comprising SEQ ID NO：The VH of amino acid sequence shown in 132 or its segment and include SEQ ID NO：139 institutes Show amino acid sequence or the VL of its segment,
(ii) comprising SEQ ID NO：The VH of amino acid sequence shown in 133 or its segment and include SEQ ID NO：138 The VL of shown amino acid sequence or its segment,
(iii) comprising SEQ ID NO：The VH of amino acid sequence shown in 134 or its segment and include SEQ ID NO：140 The VL of shown amino acid sequence or its segment,
(iv) comprising SEQ ID NO：The VH of amino acid sequence shown in 136 or its segment and include SEQ ID NO：143 The VL of shown amino acid sequence or its segment,
(v) comprising SEQ ID NO：The VH of amino acid sequence shown in 135 or its segment and include SEQ ID NO：142 institutes Show amino acid sequence or the VL of its segment,
(vi) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 or its segment and include SEQ ID NO：141 The VL of shown amino acid sequence or its segment,
(vii) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 or its segment and include SEQ ID NO：144 The VL of shown amino acid sequence or its segment,
(viii) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 or its segment and include SEQ ID NO： The VL of amino acid sequence shown in 145 or its segment and
(ix) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 or its segment and include SEQ ID NO：146 The VL of shown amino acid sequence or its segment.
Include SEQ ID NO：The VH of amino acid sequence shown in 132 can be by including SEQ ID NO：Nucleic acid sequence shown in 55 Nucleic acid encode.Include SEQ ID NO：The VH of amino acid sequence shown in 133 can be by including SEQ ID NO：Nucleic acid sequence shown in 56 Nucleic acid encode.Include SEQ ID NO：The VH of amino acid sequence shown in 134 can be by including SEQ ID NO：Nucleic acid sequence shown in 57 The nucleic acid encode of row.Include SEQ ID NO：The VH of amino acid sequence shown in 136 can be by including SEQ ID NO：Nucleic acid shown in 59 The nucleic acid encode of sequence.Include SEQ ID NO：The VH of amino acid sequence shown in 135 can be by including SEQ ID NO：Core shown in 58 The nucleic acid encode of acid sequence.Include SEQ ID NO：The VH of amino acid sequence shown in 137 can be by including SEQ ID NO：Shown in 60 The nucleic acid encode of nucleic acid sequence.
Include SEQ ID NO：The VL of amino acid sequence shown in 139 can be by including SEQ ID NO：Nucleic acid sequence shown in 62 Nucleic acid encode.Include SEQ ID NO：The VL of amino acid sequence shown in 138 can be by including SEQ ID NO：Nucleic acid sequence shown in 61 Nucleic acid encode.Include SEQ ID NO：The VL of amino acid sequence shown in 140 can be by including SEQ ID NO：Nucleic acid sequence shown in 63 The nucleic acid encode of row.Include SEQ ID NO：The VL of amino acid sequence shown in 143 can be by including SEQ ID NO：Nucleic acid shown in 66 The nucleic acid encode of sequence.Include SEQ ID NO：The VL of amino acid sequence shown in 142 can be by including SEQ ID NO：Core shown in 65 The nucleic acid encode of acid sequence.Include SEQ ID NO：The VL of amino acid sequence shown in 141 can be by including SEQ ID NO：Shown in 64 The nucleic acid encode of nucleic acid sequence.Include SEQ ID NO：The VL of amino acid sequence shown in 144 can be by including SEQ ID NO：67 institutes Show the nucleic acid encode of nucleic acid sequence.Include SEQ ID NO：The VL of amino acid sequence shown in 145 can be by including SEQ ID NO：68 The nucleic acid encode of shown nucleic acid sequence.Include SEQ ID NO：The VL of amino acid sequence shown in 146 can be by including SEQ ID NO： The nucleic acid encode of nucleic acid sequence shown in 69.
In a preferred embodiment, antibody of the invention includes VH, contains selected from embodiments below (i) extremely (vi) CDR1, CDR2 and CDR3 complementarity-determining region group：
(i)CDR1：SEQ ID NO：115 45-52 positions, CDR2：SEQ ID NO：115 70-77 positions, CDR3：SEQ ID NO：115 116-125 positions,
(ii)CDR1：SEQ ID NO：116 45-52 positions, CDR2：SEQ ID NO：116 70-77 positions, CDR3：SEQ ID NO：116 116-126 positions,
(iii)CDR1：SEQ ID NO：117 45-52 positions, CDR2：SEQ ID NO：117 70-77 positions, CDR3：SEQ ID NO：117 116-124 positions,
(iv)CDR1：SEQ ID NO：118 45-52 positions, CDR2：SEQ ID NO：118 70-77 positions, CDR3：SEQ ID NO：118 116-126 positions,
(v)CDR1：SEQ ID NO：119 44-51 positions, CDR2：SEQ ID NO：119 69-76 positions, CDR3：SEQ ID NO：119 115-125 positions,
(vi)CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions.
In a preferred embodiment, antibody of the invention includes VL, contains selected from embodiments below (i) extremely (ix) CDR1, CDR2 and CDR3 complementarity-determining region group：
(i)CDR1：SEQ ID NO：121 47-58 positions, CDR2：SEQ ID NO：121 76-78 positions, CDR3：SEQ ID NO：121 115-123 positions,
(ii)CDR1：SEQ ID NO：122 49-53 positions, CDR2：SEQ ID NO：122 71-73 positions, CDR3：SEQ ID NO：122 110-118 positions,
(iii)CDR1：SEQ ID NO：123 47-52 positions, CDR2：SEQ ID NO：123 70-72 positions, CDR3：SEQ ID NO：123 109-117 positions,
(iv)CDR1：SEQ ID NO：124 47-58 positions, CDR2：SEQ ID NO：124 76-78 positions, CDR3：SEQ ID NO：124 115-123 positions,
(v)CDR1：SEQ ID NO：125 47-58 positions, CDR2：SEQ ID NO：125 76-78 positions, CDR3：SEQ ID NO：125 115-123 positions,
(vi)CDR1：SEQ ID NO：126 47-58 positions, CDR2：SEQ ID NO：126 76-78 positions, CDR3：SEQ ID NO：126 115-122 positions,
(vii)CDR1：SEQ ID NO：127 47-58 positions, CDR2：SEQ ID NO：127 76-78 positions, CDR3：SEQ ID NO：127 115-123 positions,
(viii)CDR1：SEQ ID NO：128 47-58 positions, CDR2：SEQ ID NO：128 76-78 positions, CDR3： SEQ ID NO：128 115-123 positions and
(ix)CDR1：SEQ ID NO：129 47-52 positions, CDR2：SEQ ID NO：129 70-72 positions, CDR3：SEQ ID NO：129 109-117 positions.
In a preferred embodiment, antibody of the invention includes VH and VL, contains respectively selected from following implementation Scheme (i) to (ix) CDR1, CDR2 and CDR3 complementarity-determining region group：
(i)VH：CDR1：SEQ ID NO：115 45-52 positions, CDR2：SEQ ID NO：115 70-77 positions, CDR3： SEQ ID NO：115 116-125 positions, VL：CDR1：SEQ ID NO：122 49-53 positions, CDR2：SEQ ID NO：122 71-73, CDR3：SEQ ID NO：122 110-118 positions,
(ii)VH：CDR1：SEQ ID NO：116 45-52 positions, CDR2：SEQ ID NO：116 70-77 positions, CDR3： SEQ ID NO：116 116-126 positions, VL：CDR1：SEQ ID NO：121 47-58 positions, CDR2：SEQ ID NO：121 76-78, CDR3：SEQ ID NO：121 115-123 positions,
(iii)VH：CDR1：SEQ ID NO：117 45-52 positions, CDR2：SEQ ID NO：117 70-77 positions, CDR3： SEQ ID NO：117 116-124 positions, VL：CDR1：SEQ ID NO：123 47-52 positions, CDR2：SEQ ID NO：123 70-72, CDR3：SEQ ID NO：123 109-117 positions,
(iv)VH：CDR1：SEQ ID NO：119 44-51 positions, CDR2：SEQ ID NO：119 69-76 positions, CDR3： SEQ ID NO：119 115-125 positions, VL：CDR1：SEQ ID NO：126 47-58 positions, CDR2：SEQ ID NO：126 76-78, CDR3：SEQ ID NO：126 115-122 positions,
(v)VH：CDR1：SEQ ID NO：118 45-52 positions, CDR2：SEQ ID NO：118 70-77 positions, CDR3： SEQ ID NO：118 116-126 positions, VL：CDR1：SEQ ID NO：125 47-58 positions, CDR2：SEQ ID NO：125 76-78, CDR3：SEQ ID NO：125 115-123 positions,
(vi)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3： SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：124 47-58 positions, CDR2：SEQ ID NO：124 76-78, CDR3：SEQ ID NO：124 115-123 positions,
(vii)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3： SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：127 47-58 positions, CDR2：SEQ ID NO：127 76-78, CDR3：SEQ ID NO：127 115-123 positions,
(viii)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：128 47-58 positions, CDR2：SEQ ID NO： 128 76-78 positions, CDR3：SEQ ID NO：128 115-123 positions and
(ix)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3： SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：129 47-52 positions, CDR2：SEQ ID NO：129 70-72, CDR3：SEQ ID NO：129 109-117 positions.
In other preferred embodiments, the monoclonal antibody that antibody of the invention is preferably comprised for CLD18 is (excellent Select the monoclonal antibody described herein for CLD18) in one or more of heavy chain variable region (VH) and/or light chain variable region (VL) A complementarity-determining region (CDR) preferably at least comprising CDR3 variable regions, and preferably comprises heavy chain variable region described herein (VH) And/or one or more complementarity-determining regions (CDR) of light chain variable region (VL), preferably at least comprising CDR3 variable regions.One In a embodiment, one or more of complementarity-determining regions (CDR) are selected from CDR1, CDR2 and CDR3 as described herein complementation Property determine district's groups.In an especially preferred embodiment, the monoclonal that antibody of the invention is preferably comprised for CLD18 resists The complementation of body (the preferably monoclonal antibody described herein for CLD18) heavy chain variable region (VH) and/or light chain variable region (VL) Property determine area CDR1, CDR2 and CDR3, and preferably comprise heavy chain variable region described herein (VH) and/or light chain variable region (VL) Complementarity-determining region CDR1, CDR2 and CDR3.
In one embodiment, the sheet comprising one or more CDR, the combination of one group of CDR or CDR group as described herein Invention antibody is included intervenes the CDR of framework region (intervening framework region) together with it.It is preferred that Ground, the part also by comprising one in first and the 4th framework region or secondly at least about 50%, described 50% be first The N-terminal 50% of the C-terminal 50% of a framework region and the 4th framework region.It is constructed by the antibody of the present invention of recombinant DNA technology generation May cause variable region N-terminal or C-terminal introduce connector coding residue, the connector be introduced for convenient for clone or its His operating procedure is included including introducing connector with connecting the variable region of the present invention and other protein sequences, the other sequences Heavy chain immunoglobulin, other variable domains (such as in generation of double antibody) or protein tag.
In one embodiment, the sheet comprising one or more CDR, the combination of one group of CDR or CDR group as described herein Invention antibody includes the CDR in human antibody framework region.
Referred to herein is preferably directed to institute in its heavy chain comprising specific chain or the antibody of specific region or particular sequence All heavy chains for stating antibody include the situation of the specific chain, region or sequence.Correspondingly, this is also applied for the light of antibody Chain.
The invention further relates to nucleic acid, and it includes the genes or core for encoding antibody as described herein or part thereof (such as antibody chain) Acid sequence.The nucleic acid may be embodied in carrier, for example, plasmid, clay, virus, bacteriophage such as in genetic engineering often Advise other carriers used.The carrier may include other genes, such as allow in appropriate host cell and appropriate Under the conditions of select the marker gene of the carrier.In addition, the carrier also may include allowing code area correct table in appropriate host The expression control element reached.Such control element is known to those skilled in the art, it may include promoter, montage box and Translation initiation codon.
Preferably, nucleic acid of the invention and the above-mentioned expression control sequence for allowing to express in eukaryon or prokaryotic cell are effective Connection.The control element for ensuring to express in eukaryon or prokaryotic cell is well known to those skilled in the art.
For building the carrier of nucleic acid molecules of the present invention, structure comprising above-mentioned nucleic acid molecules, carrier being introduced correct selection Host cell, cause or realize expression method be well known in the present art.
Another aspect of the present invention relates to the host cells for including nucleic acid described herein or carrier
Other features and advantages of the present invention will be fairly obvious in described in detail below and claim.
Description of the drawings
Fig. 1 shows the immunofluorescence analysis for the HEK293 cells for having transfected the CLD18A2 being coupled with Green fluorescent dye, institute Cell is stated with using SEQ ID NO：Mice serum after 15 progress DNA immunizations is reacted, the SEQ ID NO：15 be with What auxiliary epitope (helper epitope) blended.
Fig. 2, which is shown, has transfected CLD18A2-myc (SEQ ID NO：3) HEK293 cells and the HEK293 cells of untransfected Western blot analysis, the analysis is using the anti-c-myc antibody 9E11 of Monoclonal mouse (Serotec, CRL MCA2200) It carries out.
Fig. 3 displays, which use, has transfected the Chinese hamster ovary celI of CLD18A2 and polyclonal rabbit-anti CLD18 antibody (Zymed, CRL 38- 8000) immunofluorescence analysis carried out.
Fig. 4 A and B show the doma supernatant 24H5 and 85A3 by Flow Cytometry Assay with having transiently transfected people The combination of the HEK293 cells of CLD18A2 and fluorescent marker.Fig. 4 C show doma supernatant 45C1,125E1,163E12, 166E2 and 175D10 and stable transfection people CLD18A2 and the HEK293 redyed with propidium iodide (propidium iodide) The combination of cell.
Fig. 5 shows doma supernatant 24H5 (A), 9E8 (B), 26B5 (C) and the 19B9 by Flow Cytometry Assay (D) with having transiently transfected the knot of fluorescent marker and the HEK293 cells of people CLD18A2 or CLD18A2-Myc or CLD18A2-HA It closes.
Fig. 6 A and B show by doma supernatant 37H8,43A11,45C1 and 163E12 of Flow Cytometry Assay with The stable transfection combination of the HEK293 cells of people CLD18A2 or CLD18A1.
Fig. 7 shows the immunofluorescence analysis of CLD18A2 isoform monoclonal antibody specifics 37G11, and the analysis passes through It is dyed under the conditions of natural (A, B) and paraformaldehyde fix (C, D) and has transfected CLD18A2 (A, C) and CLD18A1 (B, D) respectively HEK293 cells carry out.
Fig. 8 shows the immunofluorescence analysis of CLD18 monoclonal antibodies 26B5, and the analysis passes through natural (A, B) and more Polyformaldehyde fix under the conditions of (C, D) dye the HEK293 cells for having transfected CLD18A2 (A, C) and CLD18A1 (B, D) respectively come into Row.
Fig. 9 cell lines RT-PCR
Using the RT-PCR of CLD18A2 specific primers analysis shows that there is specific expression in 4/5 test cell system.
Figure 10 shows DAN-G cells (subclone F2) and polyclonal rabbit-anti CLD18 antibody (Zymed, CRL 38-8000) Immunofluorescence analysis.
Figure 11 shows KATO-III cells (subclone 3B94D5) and polyclonal rabbit-anti CLD18 antibody (Zymed, CRL 38- 8000) immunofluorescence analysis.
Figure 12 A show SNU-16 cells (subclone G5) and polyclonal rabbit-anti CLD18 antibody (Zymed, CRL 38-8000) Immunofluorescence analysis.Figure 12 B show KATO-III cells and the immunofluorescence analysis of monoclonal antibody of the present invention.
Figure 13 shows expression of the CLD18 in KATO-III and NUGC-4 cell surfaces, by using monoclonal antibody 61C2 It is dyed with 163E12 and then carries out flow cytometry to be analyzed.
Figure 14 people CLD18A1 (NP_057453), people CLD18A2 (NP_001002026), mouse CLD18A1 (NP_ 062789) it is compared with the protein of mouse CLD18A2 (AAL15636).
Figure 15 A and B show through doma supernatant 38G5,38H3,37G11,45C1 of flow cytometry and 163E12 respectively and transiently transfected fluorescent marker and mouse CLD18A1 or mouse CLD18A2 HEK293 cells combination.
The immunohistochemical analysis that Figure 16 are carried out using polyclonal antibody p105
The immunohistochemical staining of normal tissue subgroup (stomach, lung, marrow and prostate) confirms gastric tissue specificity (A).Also expression is detected in gastric cancer (upper row) and lung cancer (B).Only noble cells expression CLD18A2, but stem cell It does not express (C).
The immunohistochemical analysis that Figure 17 are carried out using monoclonal antibody 39F11D7
(A) detect that specific protein is expressed in normal gastric mucosa, and the every other normal structure tested is It is negative.
(B) strong CLD18A2 expression is found in gastric cancer and lung cancer.
Figure 18 are carried out using monoclonal antibody 26B5 (A), 175D10 (B), 43A11 (C), 163E12 (D) and 45C1 (E) Immunohistochemical analysis.All antibody show the strong dyeing of HEK293-CLD18A2 xenograft tumours and gastric cancer sample, and The tumour that control transfection is simulated with HEK293- is quite different.
Figure 19 compares through 85A3,28D10,24H5 or 26D12 induction for stable transfection people CLD18A2 The figure of dead cell percentage, uses Flow Cytometry Assay after the CDC of HEK293 cells.
Figure 20 be by 24H5,26D12,28D10,37G11,37H8,38G5,38H3,39F11,41C6,42E12, 43A11,44E10,47D12 or 61C2 induction are for the stable transfection adherent Chinese hamster ovary celI of people CLD18A2 or people CLD18A1 The figure of Specific cell lysis percentage after CDC, is measured by fluorescence measurement.
Figure 21 shows 75B8 (A), 28D10 (B) or the induction of 37H8 (C) concentration dependent for stable transfection people The CDC of the Chinese hamster ovary celI of CLD18A2 is measured by fluorescence measurement.
Figure 22 is shown in the HEK293-CLD18A2 that 26B5,37H8,38G5,47D12 and 61C2 in the presence of MNC are induced respectively Cell cracking.
Figure 23 is shown in the HEK293-CLD18A1 that 26B5,37H8,38G5,47D12 and 61C2 in the presence of MNC are induced respectively Cell cracking.
Figure 24 is shown in antibody pair of the present invention in the early treatment heteroplastic transplantation model using HEK293-CLD18A2 cells The inhibition of tumour growth.
Figure 25 A and B, which are shown in two kinds of early treatment heteroplastic transplantation models using HEK293-CLD18A2 cells, to be passed through Use the prolonging survival of Antybody therapy of the present invention.
Figure 26 is shown in be moved using depth treatment (advanced treatment) xenogenesis of HEK293-CLD18A2 cells It plants in model through the prolonging survival of antibody of the present invention.
Figure 27 A are shown in early treatment heteroplastic transplantation model through the Tumor growth inhibition of antibody of the present invention.Figure 27 B are shown Show in early treatment heteroplastic transplantation model through the prolonging survival of antibody of the present invention.DAN-G using endogenous expression CLD18A2 is thin Born of the same parents.
Figure 28 shows the CLD18A2mRNA expression in mouse tissue.It is studied using the RT-PCR of CLD18A2 specific primers It has been shown that, without significantly expression in all tested normal structures in addition to stomach.Analyze following normal structure：1：Small intestine, 2：Spleen, 3：Skin, 4：Stomach, 5：Lung, 6：Pancreas, 7：Lymph node, 8：Thymus gland, 9：Negative control.
Figure 29 shows the CLD18 expression in normal gastric.It is shown using the Mouse Stomach immunohistochemical analysis of CLD18 specific antibodies Conservative expression pattern is shown.Although superficial epithelium and the crypts of deeper inside (crypt) express CLD18 in its cell surface, Central neck area (central neck region) is negative for CLD18.
Figure 30 shows the h and E dyeing of mouse gastric tissue.It shows the mouse handled with 37G11 and only uses PBS The overview (A) of stomach of the control mice (C and D) of processing and details (B).
Figure 31 A and B display are using antibody of the present invention (43A11,125E1,163E12,166E2 and 175D10) to steady respectively The fluidic cell dye that the KATO-III cells of the fixed HEK293 cells for having transfected CLD18A1 and A2 and endogenous expression carry out Color.
Figure 32 shows the CDC to CLD18A2 expression cells of chimeric antibody mediation of the present invention.
Figure 33 shows the ADCC to KATO-III cells of chimeric antibody mediation of the present invention.
Specific embodiment
Antibody described herein can be the monoclonal antibody of the separation combined with epitope specificity present on CLD18.This It invents the monoclonal antibody of separation covered and includes IgA, IgG1-4, IgE, IgM and IgD antibody.In one embodiment, institute Antibody is stated as IgG1 antibody, more specifically IgG1 κ or IgG1 λ isotypes.In another embodiment, the antibody is IgG3 Antibody, more specifically IgG3 κ or IgG3 λ isotypes.In another embodiment, the antibody for IgG4 antibody more particularly For IgG4 κ or IgG4 λ isotypes.In another embodiment, the antibody is IgA1 or IgA2 antibody.In another embodiment In, the antibody is IgM antibody.
In one embodiment, the present invention relates to such antibody, are combined with the cell-specific for expressing CLD18, And preferably cell combination of (i) and expression CLD18A2, and (ii) with not expressing CLD18A2 but expressing CLD18A1 Cell combination.The antibody of the present invention preferably cell of (i) mediation killing expression CLD18A2, and (ii) does not mediate killing not It expresses CLD18A2 but expresses the cell of CLD18A1.
In another embodiment, the present invention relates to such antibody, (i) is combined with expressing the tumour cell of CLD18, (ii) not with express CLD18 normal gastric mucosa cell combination and/or (iii) not with express CLD18 non-cancer lung tissue cell With reference to.
The invention also includes such antibody, the tumour cell of (i) mediation killing expression CLD18, and (ii), which is not mediated, to be killed The normal gastric mucosa cell and/or (iii) of wound expression CLD18 do not mediate the non-cancer lung tissue cell of killing expression CLD18.
In some specific embodiments, antibody (i) of the invention on CLD18A2 with reference to being not present on CLD18A1 Epitope, preferably SEQ ID NO：21st, 22 and 23, (ii) combines the epitope being located on CLD18A2 rings 1, preferably SEQ ID NO： 28, (iii) combines the epitope being located on CLD18A2 rings 2, preferably SEQ ID NO：30, (iv), which is combined, to be located on CLD18A2 rings D3 Epitope, preferably SEQ ID NO：31, (v) combines the epitope for including CLD18A2 rings 1 and CLD18A2 rings D3, and (vi), which is combined, to be located at Non-glycosylated epitope on CLD18A2 rings D3, preferably SEQ ID NO：29 or (vii) with reference to existing in people and mouse CLD18 Epitope (be respectively SEQ ID NO：2、SEQ ID NO：8 and SEQ ID NO：35、SEQ ID NO：37).
In some particularly preferred embodiments, it is not present on CLD18A1 on antibody combination CLD18A2 of the invention Epitope.
The antibody of the present invention includes human antibody.Such antibody can be at non-human transgenic animal (such as transgenic mice) Middle generation, the transgenic animals can generate a variety of people for CLD18 by carrying out V-D-J recombinations and isotype conversion Monoclonal antibody isotype.Such transgenic animals can also be as more in generated disclosed in US 2003/0017534 The transgene rabbit of clonal antibody.
The combination of antibody and CLD18 antigen of the present invention can mediate the cell (such as tumour cell) of killing expression CLD18, such as It is mediated by activating complement system.The killing of cell to expressing CLD18 can be occurred by one or more following mechanism：Table Up to the complement-dependent cytotoxicity (CDC) of the cell of CLD18；Express the apoptosis of the cell of CLD18；Express the cell of CLD18 Effector cell's phagocytosis；Or effector cell's antibody-dependent cytotoxicity (ADCC) of the cell of expression CLD18.
In order to which the present invention is more readily understood, some terms are defined first.Other, which are defined on, is described in detail disclosed in full text.
Term defines
Term " CLD18 " refers to claudin -18, and naturally expressed including cell or cell institute that transfected CLD18 genes Variant (including CLD18A1 and CLD18A2), conformation, isoform (isoform) and the inter-species homologue of any CLD18 of expression (species homologs).Preferably, " CLD18 " refers to people CLD18, particularly CLD18A2 (SEQ ID NO：1st, 2) and/or CLD18A1(SEQ ID NO：7th, 8), more preferable CLD18A2.
It is any expressed by cell that is that term " CLD18A1 " is naturally expressed including cell or having transfected CLD18A1 genes Posttranslational modification variant, isoform and the inter-species homologue of people CLD18A1.
It is any expressed by cell that is that term " CLD18A2 " is naturally expressed including cell or having transfected CLD18A2 genes Posttranslational modification variant, isoform and the inter-species homologue of people CLD18A2.
Term " CLD18 variants " should include (i) CLD18 splice variants, (ii) CLD18 posttranslational modification variants, particularly Including the different variant of N glycosylation states, (iii) CLD18 conformational variants thereofs, particularly including CLD18- conformations -1, CLD18- structures As -2 and CLD18- conformation -3, (iv) is associated variant, (v) positioned at the free CLD18 and homotype/abnormal shape of intercellular tight junctions CLD18 cancers related variants and the non-cancer related variants of CLD18.
Term " film raft (raft) " refers to the film micro-structure rich in sphingolipid and cholesterol in cytoplasma membrane exite area Domain.Certain protein are attached to ability in these structural domains and its ability of formation " aggregation " or " lesion aggregation " can shadow Ring the function of the protein.For example, CLD18 molecules, in these structures are moved to after combining antibody of the present invention, this is in plasma membrane Generate highdensity CLD18 antigen-antibody complexes.These highdensity CLD18 antigen-antibody complexes can be in CDC processes Middle effective activation complement system.
How the membrane molecule that term " conformation " and " topology " description are integrated is located in the film of cell surface, particularly, Which region it is extracellular and thus be suitble to binding antibody.For example, CLD18 can be with three kinds of tripe systems as existing, this is likely to depend on In it whether using homopolymer or different aggressiveness as advantage or it be incorporated into supermolecule tight connecting device or " free ".These different states cause different epitopes to be suitable for binding antibody.
According to the present invention, term " disease " refers to any pathological state, including cancer, cancer shape particularly as described herein Formula.
" tumour " refers to one group of abnormal cell or tissue, they are grown by quick, uncontrolled cell Proliferation, and It continues to grow after the stimulation for originating neoformation growth stops.Tumour show the tissue of partially or completely deletion construct with And the orthofunction with normal structure, and it can be benign or malignant unique tissue block to be usually formed.
" transfer " refers to other positions that cancer cell travels to body from its initial site.The formation of transfer is extremely complex Process, be detached from from primary tumo(u)r dependent on malignant cell, invade extracellular matrix, through interior basement membrane with enter body cavity and blood It manages and infiltrates target organ after being transported by blood thereafter.Finally, new tumour depends on angiogenesis in the growth of target area.Tumour turns Even occur after primary tumo(u)r is cut off moved through normal, because tumour cell or component may remain and develop metastatic potential. In one embodiment, term of the invention " transfer " refers to " far-end transfer ", this refers to far from primary tumo(u)r and regional nodes The transfer of system.
The treatment of term " treatment disease " including disease or its symptom, the shortening of duration, improvement, prevent, delay or It is inhibited to develop or deteriorates or delay its breaking-out.
According to the present invention, sample can be according to the available any sample of the present invention, and particularly biological sample such as organizes sample Product (including body fluid) and/or cell sample, and can obtain in a usual manner, for example, by organize biopsy (including the biopsy that drills) and It is obtained by collecting blood, bronchial perfusate, phlegm, urine, excreta or other body fluid.According to the present invention, term " biological sample Product " further include the fraction of biological sample.
Term " antibody " refers to comprising at least two weight (H) chains and two light (L) chains that are interconnected by disulfide bond or its antigen The glycoprotein of binding fragment.Term " antibody " further includes all recombinant forms of antibody (particularly antibody described herein), such as The antibody expressed in prokaryotic cell, not glycosylated antibody and with the antibody fragment of antigen binding and derivative described below Object.Each heavy chain is made of heavy chain variable region (being abbreviated as VH herein) and heavy chain constant region.Every light chain is by light chain variable region (this Text is abbreviated as VL) and constant region of light chain composition.VH and VL areas can be further subdivided into referred to as the high of complementarity-determining region (CDR) and become Area, they are dispersed in the referred to as more conservative region of framework region (FR).Every VH and VL is made of three CDR and four FR, from Aminoterminal arranges in the following order to c-terminus：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4.Heavy chain and light chain it is variable Contain the binding structural domain with antigen interactions in area.The constant region of antibody can mediate the immunoglobulin and host tissue or because The combination of son, the host tissue or the factor include various kinds of cell (such as effector cell) and the classical complement system of immune system First composition (C1q).
Term " humanized antibody " refers to the molecule with the antigen binding site substantially from non-human species' immunoglobulin, Wherein described remaining immunoglobulin structure of molecule is structure and/or sequence based on human immunoglobulin(HIg).The antigen knot Conjunction site may include being fused to the complete variable domains or only suitable comprising being transplanted in variable domains in constant domain When the complementarity-determining region (CDR) of framework region.Antigen binding site can be wild type or by one or more amino Acid is replaced and is modified, such as modified with more close with human immunoglobulin(HIg).Some form of humanized antibody retains Whole CDR sequences (such as humanization mouse antibodies containing all six CDR from mouse antibodies).Other forms have The CDR that one or more is changed for original antibodies.
Term " chimeric antibody " refers to such antibody, wherein a part for each heavy chain and light-chain amino acid sequence is with coming from Corresponding amino acid sequence homologous or belong to specific classification in the antibody of particular species, and remaining section of the chain then with separately Corresponding sequence in one species is homologous.Usually, the variable region of light chain and heavy chain is simulated from mammalian species The variable region of antibody, and constant portion is then homologous with the antibody sequence from another species.One of this chimeric versions thereof is apparent Advantage be can be used the B cell that is easily obtained or hybridoma from inhuman host organism from the source being currently known easily Variable region is generated, and constant region in combination comes from such as people's cell prepared product.The variable region has easily prepared excellent Point, and specificity is not influenced by source, and since constant region is the mankind, which causes people experimenter in injection The possibility of immune response will come from lower during non-people source than constant region.However, define specific example without being limited thereto.
" antigen-binding portion thereof " (or being referred to as " binding site ") of terms used herein antibody, which refers to, retains spy in antibody The opposite sex combines one or more segments of the ability of antigen.It has been shown that the antigen binding function of antibody can pass through full length antibody Segment realize.Belong to the example of binding fragment that " antigen-binding portion thereof " of antibody is covered and include (i) Fab segments, by The monovalence segment of VL, VH, CL and CH structural domain composition；(ii)F(ab′)2Segment, comprising being connected by disulfide bond at hinge area Two Fab segments bivalent fragment；(iii) the Fd segments being made of VH and CH structural domains；(iv) by the VL of antibody single armed and The Fv segments of VH structural domains composition；(v) dAb segments (Ward etc., (1989) Nature 341 being made of VH structural domains：544- 546)；(vi) independent complementarity-determining region (CDR) and (vii) optionally connected by synthetic linker two or more The combination of a independent CDR.In addition, although two structural domain VL and VH of Fv segments by individual gene code, they can It is linked together using recombination method by synthetic linker, the connector makes them become single protein chain, wherein VL and VH It matches to form monovalent molecules (referred to as scFv (scFv), refering to such as Bird etc. (1988) Science 242 in area：423-426 and Huston etc. (1988) Proc.Natl.Acad.Sci.USA 85：5879-5883).Such single-chain antibody, which also aims to, to be included in In " antigen-binding portion thereof " of term antibody.Another example is comprising the binding structural domain immunoglobulin fusion egg with lower part In vain：(i) the integrated structure domain polypeptide merged with immunoglobulin hinge region polypeptide, the immune globulin that (ii) is merged with the hinge area The heavy chain immunoglobulin CH3 constant regions of Bai Chonglian CH2 constant regions and (iii) and CH2 constant domains.The integrated structure Domain polypeptide can be heavy chain variable region or light chain variable region.The binding structural domain domain-immunoglobulin fusion proteins are in US 2003/ It is further described in 0118592 and US 2003/0133939.Well known to a person skilled in the art normal for these antibody fragment uses Rule technology obtains, and the segment is screened in a manner of identical with complete antibody.
Term " epitope " refers to the protein determinant combined with antibody, and wherein term " with reference to " preferably refers to herein Specific binding.Epitope is usually made of, and usually have the chemically active surface group of molecule (such as amino acid or carbohydrate side chain) Specific three-dimensional structural feature and specific charge characteristic.Difference lies in deposited in denaturant for comformational epitope and non-conformational epitope It is lower the former lose combination, and the latter is quite different.
By only at least two Protein primary sequences on terms used herein " discontinuous epi-position " finger protein matter antigen The comformational epitope of vertical region composition.
Term " bispecific molecule " is intended to include any reagent of the tool there are two types of different binding specificities, as protein, Peptide or protein or peptide complexes.For example, the molecule can with (a) cell surface antigen and the Fc on (b) effector cell surface by Body combines or interaction.Term " multispecific molecule " or " heterospecific molecule " are intended to include having more than two kinds of differences Any reagent of binding specificity, such as protein, peptide or protein or peptide complexes.For example, for example, the molecule can be with (a) Cell surface antigen, the Fc receptors on (b) effector cell surface and (c) at least one other components combine or interact.Cause This, the present invention is included but are not limited to for CLD18 and for the double special of other targets (the Fc receptors on such as effector cell) Property, tri-specific, four specificity and other multispecific molecules.Term " bispecific antibody " further includes double antibody.Double antibody It is bivalent, bispecific antibodies, wherein VH and VL structural domains are expressed in single polypeptide chain, but the connector used is too short, so that Two structural domains on same chain is not allowed to match, so as to which structural domain be forced to match and generate with the complementary domain on another chain Two antigen binding sites (refer to such as Holliger, P., wait (1993) Proc.Natl.Acad.Sci.USA 90：6444- 6448；Poljak, R.J. wait (1994) Structure 2：1121-1123).
The invention also includes the derivatives of antibody described herein.Term " antibody derivatives " refers to any modification shape of antibody Formula, such as antibody and another reagent or the conjugate of antibody.The specific reproduction of antibody " coming from " used herein in the case where there It is sequence：I.e. if antibody derives from a system, and its by immune animal or by screening immunoglobulin gene libraries In selected antibody amino acid sequence and the germline immunoglobulin coded by said gene amino acid sequence have at least 90%, More preferably at least 95%, even more desirably at least 96%, 97%, 98% or 99% homogeneity.In general, from specific system genitale The antibody of sequence will show not more than 10 amino acid differences with the amino acid sequence of the germline immunoglobulin coded by said gene It is different, more preferably no more than 5 amino acid of differences or even more preferably not more than 4,3,2 or 1 amino acid of differences.
Terms used herein " heterologous antibody " refer to two or more antibody to link together, its derivative or antigen Combined area, wherein at least two have different specificity.The Fc of these different specificity including pairing effect cell surface by The binding specificity of body and to the antigen or the binding specificity of epitope on target cell (such as tumour cell).
Antibody as described herein can be human antibody.Terms used herein " human antibody " are intended to include having from life Grow is the variable region of immunoglobulin sequences and the antibody of constant region.The human antibody of the present invention may include being exempted from by people's system genitale The amino acid residue of epidemic disease globin sequence coding (such as is dashed forward by external random mutagenesis or direct mutagenesis or by internal body cell Become the mutation introduced).
Terms used herein " monoclonal antibody " refer to the antibody molecule prepared product of unimolecule composition.Monoclonal antibody is shown To the single binding specificity and compatibility of defined epitope.In one embodiment, monoclonal antibody is generated by hybridoma, institute It states hybridoma and includes the B cell derived from non-human animal (such as mouse) merged with immortalized cells.
Terms used herein " recombinant antibodies " by recombinant means including being prepared, expression, is generated or is detached all anti- Body, such as animal (such as mouse) or the hybridoma prepared therefrom of (a) from immunoglobulin gene for transgenosis or transfection chromosome The antibody of middle separation, (b) from inverted to express the antibody detached in the host cell of antibody (such as transfectoma), (c) is from recombination Combinatorial antibody library in the antibody that detaches with (d) by being related to the appointing into DNA sequence dna by Ig gene sequences montage The antibody that his means prepare, express, generate or detach.
Recombination eukaryotic host cell of the terms used herein " transfectoma " including expressing antibody, such as Chinese hamster ovary celI, NS/0 Cell, HEK293 cells, HEK293T cells, plant cell or fungi (including yeast) cell.
The genetically modified organism that terms used herein " heterologous antibody " just generate this antibody is defined.The term refers to this The antibody of sample, amino acid sequence or nucleic acid sequence encoding correspond to the biology for not including the genetically modified organism, and usually come From the species different from the genetically modified organism.
Terms used herein " hetero hybrid antibody (heterohybrid antibody) " refer to biological source difference Light chain and heavy chain antibody.For example, it is hetero hybrid antibody with the antibody of people's heavy chain being connect with mouse light chain.
Antibody described herein is preferably what is detached.Terms used herein " antibody of separation ", which are intended to refer to, is substantially free of antigen The antibody of other antibody of specific difference is (for example, the separation antibody of specific binding CLD18 is substantially free of specific binding and removes CLD18 is with the antibody of exoantigen).However, the antibody of separation combined with epitope, isoform or the variant specificity of people CLD18 There can be cross reactivity with other related antigens (such as antigen from other species, such as CLD18 inter-species homologue).In addition, The antibody of separation can be substantially free of other cell materials and/or chemical substance.In one embodiment of the invention, " separation " combination of monoclonal antibody refers to not homospecificity and combine the antibody in clear and definite determined combination object.
According to the present invention, term " with reference to " preferably refers to " specific binding ".Terms used herein " specific binding " refer to The antibody combined with predetermined antigens.In general, the affinity that antibody combines corresponds to about 1 × 10-7M or smaller KD, and with it is pre- Determine the K corresponding to the affinity of antigen bindingDHeterogenetic antigen (such as BSA, the junket egg in addition to the predetermined antigens are combined than it In vain) or low at least two orders of magnitude of the affinity of closely related antigen.
Terms used herein " KD" (M) be intended to refer to the Dissociation equilibrium constant of specific antibody-antigene interaction.
Terms used herein " isotype " refer to the encoded antibody isotype of weight chain constant area gene (such as IgM or IgG1).
Terms used herein " isotype conversion " refer to the classification of antibody or isotype becomes another Ig by an Ig classification The phenomenon that classification.
During using Mr. Yu's object, terms used herein " naturally occurring " refer to the object and are found in nature this thing It is real.Can be from the biology (including virus) that nature source detaches for example, being present in, and it is not intended for people in the lab The polypeptide or polynucleotide sequence of work modification are naturally occurring.
Terms used herein " rearrangement " refer to the configuration of heavy chain or light chain immunoglobulins locus, and wherein V sections are being compiled The position of D-J or J sections is located close in the conformation of the basic complete VH or VL structural domains of code respectively.The immunoglobulin of rearrangement (antibody) locus can be identified by the comparison with system genitale DNA, and the locus of rearrangement is by seven at least one recombination The aggressiveness homology element of aggressiveness/nine.
When being related to V sections, terms used herein " not resetting " or " germline configuration " refer to V sections and are not reassembled as and D Or the adjacent conformation of J sections.
Terms used herein " nucleic acid molecules " are intended to include DNA molecular and RNA molecule.Nucleic acid molecules can be it is single-stranded or Double-strand, but preferably double-stranded DNA.
Nucleic acid of the present invention is preferably what is detached.According to the present invention, it is (i) that term " nucleic acid of separation ", which refers to the nucleic acid, It expands, such as is expanded by PCR (PCR) in vitro, (ii) is generated by cloning recombination, (iii) purifying , such as synthesized by digestion and gel electrophoresis classification separation or (iv), such as pass through chemical synthesis.The nucleic acid of separation It is the nucleic acid that can be operated by recombinant DNA technology.
According to the present invention, nucleic acid individualism or with other Nucleic acid combinations exist, other described nucleic acid can be it is homologous or Heterologous.In some preferred embodiments, nucleic acid is connect with expression control sequence functionality, the expression control sequence pair Can be homologous or heterologous in the nucleic acid.Term " homologous " refers to the core also natively being connect with expression control sequence functionality Acid, it is not natively to be connect with the expression control sequence that term " heterologous ", which refers to nucleic acid,.
If RNA and/or the nucleic acid of protein or peptide are expressed with expression control sequence with the expression or transcription of the nucleic acid The mode under the control or influence of the expression control sequence is in be covalently attached each other, then they each other " functionality " even It connects.If nucleic acid functional protein to be translated, and it carries the expression control sequence being connect with coded sequence functionality, The induction of the expression control sequence causes the transcription of the nucleic acid, without leading to the frameshit in coded sequence or leading to the sequence Row cannot translate into required protein or peptide.
According to the present invention, term " expression control sequence " is including promoter, ribosome bind site, enhancer and adjusts base Because of other control elements of transcription or mRNA translations.In some specific embodiments of the present invention, the expression control sequence It can be regulated.The precise structure of expression control sequence can change, but generally comprise according to the function of species or cell type Respectively participate in transcription and translation starting 5 ' non-transcribed sequences and 5 ' and 3 ' non-translated sequences, such as TATA boxes, cap sequence, CAAT sequences etc..More specifically, 5 ' non-transcribed expression control sequencs include promoter region, promoter region includes to transcribe control The promoter sequence of functionality connection nucleic acid.Expression control sequence may also include enhancer sequence or upstream activator sequence.
According to the present invention, term " promoter " or " promoter region " refer to such nucleic acid sequence：It is located at expressed nucleic acid The upstream (5 ') of sequence, and control the expression of the sequence by providing identification and the binding site of RNA polymerase." promoter Area " may include other identifications and binding site for participating in other factors that genetic transcription is adjusted.Promoter can control protokaryon Or the transcription of eukaryotic gene.In addition, promoter can be " induction type ", can response in inducer originate transcription or if Transcription can be then " composing type " by the control of inducer.If derivant is not present, under inducible promoter control Gene is not expressed or to express degree very low.There are during inducer, which starts to transcribe or transcriptional level improves.It is general and Speech, this is mediated by the combination of idiosyncratic transcription factor.
Currently preferred promoter includes the promoter, people U6RNA promoters, CMV for SP6, T3 and T7 polymerase Promoter and its artificial hybrid's promoter (such as CMV), wherein start moiety and other cell proteins (such as people GAPDH, it is sweet Oily aldehyde -3- phosphate dehydrogenases) gene promoter certain partial fusion, and comprising or not comprising additional introne.
According to the present invention, term " expression " is used with its most common meaning, including generate RNA or RNA and protein/ Peptide.Further include the part expression of nucleic acid.In addition, expression can be carried out instantaneously or stablize progress.
In a preferred embodiment, nucleic acid molecules of the invention are present in carrier, also with control when appropriate The promoter of the expression of nucleic acid.Term " carrier " is used herein with its most common meaning, including being used for any of nucleic acid Intermediate carrier, the carrier enable the nucleic acid to be for example introduced into protokaryon and/or eukaryocyte, and it is appropriate when be integrated into Genome.This kind of carrier is replicated and/or is expressed preferably in the cell.Terms used herein " plasmid " refer generally to can be independently of The extrachromosomal inhereditary material that chromosomal DNA is replicated, usually cyclic DNA duplex.
With regard to for for expressing the carrier of antibody, heavy chain of antibody and light chain are present in carrier type in different carriers and again The carrier type that chain and light chain are present in identical carrier can be used.
The introduction provided for specific nucleic acid and amino acid sequence (as shown in sequence table) herein is interpreted as also relating to And the modification to the particular sequence, the sequence generated with the particular sequence function equivalence, such as display of modifying is with being somebody's turn to do Coded by the amino acid sequence of the same or similar characteristic of specific amino acid sequence and coding display and the specific nucleic acid sequence The nucleic acid sequence of the amino acid sequence of the same or similar characteristic of amino acid sequence.One important characteristic is to retain antibody and its The combination of target or the effector function for maintaining antibody.Preferably, the sequence modified for particular sequence for Retain the antibody and the combination of CLD18 during the particular sequence changed in antibody, and it is as described herein preferably to retain the antibody Function, such as CDC mediation splitting action or ADCC mediation splitting action.
It is tied it will be appreciated by those skilled in the art that can particularly modify CDR, hypervariable region and the sequence of variable region without losing Close the ability of CLD18.For example, CDR region can or very high homology identical with region described herein." very high homology " is considered can be with 1 to 5, preferably 1 to 4 such as 1 to 3 or 1 or 2 replacement is generated in CDR.In addition, hypervariable region and variable region can be modified, so as to Significant homology is shown with region specifically disclosed herein.
It should be understood that specific nucleic acid as described herein further includes to optimize the codon in particular host cell or biology Using and the nucleic acid modified.The difference that codon uses between biology can cause a variety of asking about allogeneic gene expression Topic.By change one or more of original series nucleotide carry out codon optimization can be in the same of the nucleic acid to be expressed Cause the optimization of the optimization of expression of nucleic acid, particularly translation efficiency in source or heterologous host.If for example, it need to be made according to the present invention With the nucleic acid from people and encoding antibody constant region and/or framework region, such as be used to prepare chimeric or humanized antibody, then in order to Optimization codon uses and to modify the nucleic acid may be preferred, if particularly the nucleic acid (optionally with heterologous core Nucleic acid fusion of the acid such as from other biologies described herein) it need to be in different from the other biological of people (such as mouse or hamster) cell In the case of expression.For example, it is separately encoded nucleic acid sequence (such as SEQ ID NO of people's light chain and heavy chain constant region：40 and 45 sequence Row) can through modification, with comprising it is one or more, preferably at least 1,2,3,4,5,10,15,20 and preferably up to 10,15,20, 25th, 30,50,70 or 100 or more nucleotide are replaced, and the replacement causes the optimization that codon uses, but do not cause amino The change of acid sequence.Such nucleotide, which is replaced, preferably to be referred to respectively by SEQ ID NO：Nucleotide in 40 and 45, which replaces with, not to be drawn The modified counterpart that coded amino acid sequence changes is played, the nucleotide is respectively selected from hereafter SEQ ID NO：40 and 45 Comparison described in replacement；Or refer to and be separately encoded corresponding position in other nucleic acid sequences of people's light chain and heavy chain constant region It is corresponding to replace.Preferably, hereafter SEQ ID NO are realized respectively in the nucleic acid sequence of encoding human light chain and heavy chain constant region：40 Whole replacements shown in comparison with 45, described be replaced do not cause being corresponded to through modification for coded amino acid sequence change Object.
SEQ ID NO：40 and SEQ ID NO：147 comparison：
SEQ ID NO：45 and SEQ ID NO：149 comparison：
In addition, according to the present invention, it may be necessary to modify the sequence of amino acid sequence described herein, particularly people's heavy chain constant region Row, so that the sequence is suitable for desired allograft, such as the allograft being found in white people.Such modification is excellent Choosing is selected from following SEQ ID NO：The replacement of corresponding position in amino acid substitution or other people heavy chain constant region in 46： K93R, D235E and L237M.Preferably, all these modifications are all contained in the amino acid sequence of people's heavy chain constant region.
According to the present invention, term " corresponding position " refers to the core to correspond to each other in the sequence alignment of two nucleic acid or protein sequence Thuja acid or amino acid residue.
Preferably, specific nucleic acid sequence described herein and the nucleic acid sequence modified to the specific nucleic acid sequence it Between homogeneity be at least 70%, preferably at least 75%, more preferably at least 80%, even more desirably at least 90% or optimal Choosing at least 95%, 96%, 97%, 98% or 99%.Preferably, two sequences can hybridize and form stable duplex each other, The hybridization preferably carries out under the condition (stringent condition) for allowing polynucleotides specific hybrid.Stringent condition is described in for example Molecular Cloning：The editors such as A Laboratory Manual, J.Sambrook, the second edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols The editors such as in Molecular Biology, F.M.Ausubel, John Wiley ＆ Sons, Inc., New York, refer to example In hybridization buffer (3.5 × SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% ox blood such as at 65 DEG C Pure albumen, 2.5mM NaH2PO4(pH 7), 0.5%SDS, 2mM EDTA) in hybridize.SSC is 0.15M sodium chloride/0.15M lemons Lemon acid sodium, pH 7.After hybridization, the film of DNA has been shifted in washing, such as washs in 2 × SSC at room temperature, then up to 68 It is washed in 0.1-0.5 × SSC/0.1 × SDS at DEG C.
Preferably, specific amino acid sequence described herein and the amino acid modified the specific amino acid sequence The similitude (preferably homogeneity) of (such as show the amino acid sequence of notable homogeneity between) is at least 70% between sequence, excellent Choosing at least 80%, even more desirably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%.
All above-mentioned modification sequences are all within the scope of the present invention.
" sequence similarity " refers to the percentage of amino acid that identical or display conservative amino acid is replaced.Two polypeptides or nucleic acid " sequence identity " between sequence refers to the percentage of identical amino acid between the sequence or nucleotide.
" percentage identity " obtains after optimal comparison, this percentage is entirely two sequences on statistical significance Difference random distribution between row is over the entire length thereof.Sequence between two nucleotide or amino acid sequence compare by its into Compare its sequence after row optimal comparison routinely to carry out, the comparison is carried out by section or " comparing window ", with identification and Compare the similar regional area of sequence.It, can also be by set about for the sequence optimal comparison that compares other than generating by hand Section generates：Smith and Waterman, 1981, Ads App.Math.2,482 local homology algorithm, Neddleman and Wunsch, 1970, J.Mol.Biol.48,443 local homology algorithm, Pearson and Lipman, 1988, Proc.Natl The similarity retrieval method of Acad.Sci.USA 85,2444 or using these algorithms computer program (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA, in winconsin Genetics Software packet, genetic computation group, 575 Science Drive, Madison, Wis.).
Percentage identity is calculated by the following method：Identical positional number between two sequences that measure is compared, is used The number divided by the positional number being compared, 100 are multiplied by with result, so as to obtain the percentage identity between the two sequences.
" conservative replacement " can be based on as involved residue polarity, charge, solubility, hydrophobicity, hydrophily and/or Similitude in terms of amphipathic characteristic carries out.For example, (a) nonpolarity (hydrophobic) amino acid includes alanine, leucine, different bright ammonia Acid, valine, proline, phenylalanine, tryptophan and methionine；(b) polar neutral amino acid includes glycine, silk ammonia Acid, threonine, cysteine, tyrosine, asparagine and glutamine；(c) positively charged (alkalinity) amino acid include arginine, Lysine and histidine；(d) negatively charged (acidity) amino acid includes aspartic acid and glutamic acid.Replacement generally can be in (a)-(d) It is carried out in group.In addition, glycine and proline, which can be based on it, destroys the ability of [α] spiral and substituted for one another.It can be at following group It is middle carry out some preferably replace：(i) S and T；(ii) P and G；And (iii) A, V, L and I.In known genetic code and Under conditions of recombination and synthetic DNA technology, technical staff can easily build the DNA of coding conservative amino acid variant.
The present invention, which is included in Fc areas, to be modified to change the antibody of the antibody function or pharmacokinetic properties.This The modification of sample can cause C1q to combine reduction or raising with ADCC with reference to the reduction with CDC or raising or Fc γ R.For example, It can be replaced in one or more amino acid residues of heavy chain constant region, so as to cause effector compared with modifying antibody The change of function retains the ability with reference to antigen simultaneously, refering to US 5,624,821 and US 5,648,260.
The salvage receptor epitope of Ig constant domains or Ig sample constant domains can be modified so that the molecule has not included Whole CH2 structural domains or complete Ig Fc areas, so as to improve the Half-life in vivo of antibody, refering to US 6,121,022 and US 6, 194,551.Half-life in vivo can also be improved by generating mutation in Fc areas, such as replace 252 bright with threonine Propylhomoserin, the serine that 254 are replaced with threonine replace the phenylalanine of 256 with threonine, refering to US 6,277, 375。
It is furthermore possible to vary the glycosylation pattern of antibody, to change the effector function of the antibody.It for example, can be not It adds in the transfectoma of fucose being commonly attached on 297, Fc areas Asn and expresses antibody, to enhance the Fc areas to Fc receptors Affinity, this raising that will then cause antibody A DCC in the presence of NK cells, refering to Shield etc. (2002) JBC, 277： 26733.It is furthermore possible to vary it glycosylates to change CDC.
Alternatively, in another embodiment, mutation is randomly incorporated into all or part of anti-CLD18 antibody coding sequences, Such as introduced by saturation mutagenesis, it can be directed to and combine activity to obtaining screening through modifying anti-CLD18 antibody.
Terms used herein " recombinant host cell " (or being referred to as " host cell "), which are intended to refer to, wherein introduces recombination The cell of expression vector.It should be understood that such term is not only intended to refer to specific subject cell, and refer to these cells Offspring.Since mutation or environment influence and certain changes may occur in subsequent generation, these offsprings in fact may not be with Parental cell is identical, but is included in the range of terms used herein " host cell ".For example, recombinant host cell includes Transfectoma, such as Chinese hamster ovary celI, NS/0 cells and lymphocyte.
Terms used herein " subject " include anyone or non-human animal.Term " non-human animal " includes all vertebras Animal, such as mammal and nonmammalian, such as non-human primates, sheep, dog, ox, chicken, amphibian, reptile Deng.
Term " transgenic animals " refers to such animal：One or more transgenosis (preferably heavy chains are included in its genome And/or chain transgene) or transfection chromosome (the natural gene group DNA for integrating or not being integrated into the animal), and preferably can Enough express the transgenosis.For example, transgenic mice can have people's chain transgene and people's heavy chain transgene or the transfection of people's heavy chain Colour solid resists so as to which the mouse generates the anti-CLD18 of people when carrying out immune with the cell of CLD18 antigens and/or expression CLD18 Body.People's heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as the situation of transgenic mice, such as HuMAb mouse such as HCo7 or HCo12 mouse or people's heavy chain transgene can maintain outside chromosome, as WO 02/ The situation of 43478 transfection chromosome (such as the KM) mouse.Such transgenosis and transchromosomic mice may can pass through Carry out V-D-J recombination and isotype conversion and generate for CLD18 a variety of human monoclonal antibodies isotypes (such as IgG, IgA and/ Or IgE).
The mechanism of action of mAb
Although provided hereinafter in terms of the mechanism of antibody curative effect behind of the present invention some consideration, this it is not considered that It is to limit the invention in any way.
Antibody described herein preferably interacts with the component of immune system, is preferably occurred by ADCC or CDC.The present invention Antibody can also be used to targeting load (such as radioactive isotope, drug or toxin) with direct killing tumour cell or can be with Conventional chemotherapeutics collaboration uses, and attacks tumour by complementarity mechanism of action, the mechanism may include due to chemotherapy To the cytotoxicity side effects of T lymphocytes and impaired anti-tumor immune response.
Cytotoxicity (the Antibody-dependent cell-mediated of antibody dependent cellular mediation cytotoxicity).ADCC describes the cellkilling capacity of effector cell described herein particularly lymphocyte, this is preferably needed Target cell is wanted to be marked by antibody.
Antigens and antibody Fc domain of the ADCC preferably on antibody combination tumour cell combine immune effector cell table Occur during Fc receptors (FcR) in face.Several Fc receptor families are identified, specific cell mass is characteristically expressed determining Fc receptors.ADCC can be regarded as directly inducing the mechanism of different degrees of direct tumor destruction, and the destruction causes the antigen to be in It passs and induces tumour directive property t cell response.Preferably, the Immune inducing in vivo of ADCC by cause be directed toward tumour t cell response and Antibody response from host.
Complement-dependent cytotoxicity (Complement-dependent cytotoxicity).CDC is antibody guidance Another cell killing method.For complement activation, IgM is most effective isotype.IgG1 and IgG3 lives by classical complement Change approach instructs CDC aspects also all highly effective.Preferably, in this cascade, the formation of antigen-antibody complex causes tightly Neighbour participates in antibody molecule (such as IgG molecules) CHMultiple C1q binding sites of 2 structural domains are exposed that (C1q is the three of complement C1 One of kind subfraction).Preferably, the C1q-IgG interactions of previous low-affinity are transformed into height by exposed C1q binding sites Affinity interacts, and this triggers a series of cascade for including other complement proteins, and causes effector cell's chemotactic/activator The proteolysis release of C3a and C5a.Preferably, which ultimately forms membrane attack complex, it is generated in cell membrane Hole, is conducive to water and solute freely navigates within intraor extracellular.
The generation of antibody
The antibody of the present invention can be generated by multiple technologies, including conventional monoclonal antibody method, such as Kohler and Milstein, Nature 256：The standard somatic cell hybridization technology of 495 (1975).Although preferred somatic hybridization side in principle Case, but can also utilize and generate the other technologies of monoclonal antibody, for example, the virus of bone-marrow-derived lymphocyte or neoplastic transformation or Use the display technique of bacteriophage of library of antibody genes.
The preferred animal system for being used to prepare the hybridoma of secrete monoclonal antibody is mouse system.Hybridoma in mouse Generation is highly developed scheme.Separation for fusion immune spleen cell immune operation and technology by this field public affairs Know.Fusion partner (fusion partner) (such as murine myeloma cell) and mixing operation are also known.
Other animal systems for being used to prepare the hybridoma of secrete monoclonal antibody are rat and rabbit system (such as Spieker- Polet etc., Proc.Natl.Acad.Sci.U.S.A.92：9348 (1995) are described, also see Rossi etc., Am.J.Clin.Pathol.124：295(2005)).
In another preferred embodiment, it can be used for the human monoclonal antibodies of CLD18 with groups of people's siberian crabapple The transgenosis or transchromosomic mice of system rather than mouse system generate.These transgenosis and transchromosomic mice include difference The referred to as mouse of HuMAb mouse and KM mouse will be collectively referred to herein as " transgenic mice ".It can be according in WO2004 035607 Human antibody is generated to being described in detail in these transgenic mices for CD20.
Another strategy for generating monoclonal antibody is to be directly separated coding from the lymphocyte for generating determining tactful antibody The gene of antibody, refering to such as Babcock etc., 1996；A novel strategy for generating monoclonal Antibodies from single, isolated lymphocytes producing antibodies of defined strategy.The details of recombinant antibodies engineering also sees Welschof and Kraus, Recombinant antibodes for Cancer therapy ISBN-0-89603-918-8 and Benny K.C.Lo Antibody Engineering ISBN 1- 58829-092-1。
It is immune
In order to generate the antibody for being directed to CLD18, peptide, recombination table can be conjugated with the carrier from CLD18 sequences as described The CLD18 antigens or the enriched preparation of its segment that reach and/or the cell of CLD18 is expressed mouse is immunized.It is compiled alternatively, can use DNA (such as SEQ ID NO of code overall length people CLD18：1) or its segment (is particularly SEQ ID NO：15th, 17 and 19 segment) exempt from Epidemic disease mouse.The CLD18 antigen preparations for using purifying or enrichment carry out it is immune do not generate the event of antibody, can also use Mouse is immunized in the cell (such as cell line) for expressing CLD18, to promote immune response.
It can be with by the way that the blood plasma of blood acquisition and blood serum sample be taken to be monitored in the whole process of immunization protocol after tail vein or socket of the eye Immune response.The enough mouse of anti-CLD18 immunoglobulins potency can be merged.It can be 3 before putting to death and extracing spleen It is intraperitoneal or intravenously carry out booster immunization to mouse with the cell of expression CLD18, to improve the miscellaneous of secreting specificity antibody Hand over the ratio of knurl.Generate the hybridoma of production monoclonal antibody
In order to generate production CLD18 monoclonal antibodies hybridoma, can from immune mouse separating Morr. cell and lymph node Cell, and merged with appropriate immortalized cell line (such as mouse myeloma cell line).Then antigen-specific antibodies can be directed to The obtained hybridoma of generation screening.Then the hybridoma of secretory antibody can be screened to single hole by ELISA.By using The immunofluorescence and facs analysis that CLD18 expression cells carry out, can identify the antibody for having specificity to CLD18.It can will divide Secrete the hybridoma of antibody bed board again, screen again, and if anti-CLD18 monoclonal antibodies be still it is positive if can pass through Limiting dilution is subcloned.Then the subclone that can be stablized in vitro culture is used for generating antibody in tissue culture medium (TCM) Characterization.
Generate the transfectoma of production monoclonal antibody
The antibody of the present invention can also generate, such as use recombinant DNA well known in the art in host cell transfectoma The combination of technology and Gene transfer techniques carries out (Morrison, S. (1985) Science 229：1202).
For example, in one embodiment, target gene (such as antibody gene) can be connected into expression vector such as eukaryon table Up in plasmid, such as by GS gene expression systems described in WO 87/04462, WO 89/01036 and EP 338 841 or originally Other expression systems known to field carry out.Can by band Cloning Human Immunoglobulin Genes plasmid purification introduce eukaryotic host cell In, such as Chinese hamster ovary celI, NS/0 cells, HEK293T cells or HEK293 cells or other eukaryocytes, as from plant Cell, fungi or yeast cells.Method for introducing these genes can be this field it has been described that method, as electricity is worn Hole, lipofectine, lipofectamine or other.After these antibody genes are introduced host cell, it can identify and select Select the cell for expressing the antibody.These cells represent its expression amplifiable thereafter and expand scale to produce the transfection of antibody Knurl.Recombinant antibodies can be detached and be purified from these culture supernatants and/or cell.Alternatively, the antibody gene of clone can be with It is expressed in other expression systems, including prokaryotic cell such as microorganism, such as Escherichia coli.In addition, antibody can also be inhuman It is generated in transgenic animals, such as generates in the breast of sheep and rabbit or in egg or generate in transgenic plants, joined It reads such as Verma, R., waits (1998) J.Immunol.Meth.216：165-181；Pollock is waited (1999) J.Immunol.Meth.231：147-157 and Fischer, R. wait (1999) Biol.Chem.380：825-839.
Use partial antibody sequences expression complete antibody (i.e. humanization and be fitted into)
A) it is fitted into
When being marked with toxin or radioactive isotope, mouse monoclonal antibody can be used as therapeutic antibodies in people. Unlabelled mouse antibodies have high immunogenicity in repeated application in people, and curative effect is caused to reduce.Main immunogenicity It is mediated by heavy chain constant region.If carrying out chimeric or humanized to each antibody, immunogenicity of the mouse antibodies in people can To reduce or avoid completely.Chimeric antibody is antibody of the different piece from different animals species, such as with from mouse The variable region of antibody and the antibody of human immunoglobulin(HIg) constant region.Antibody it is chimeric by by mouse antibody light chain and light chain variable Area is joined together to realize (such as Kraus, Methods in Molecular Biology with people's heavy chain and constant region of light chain Described in series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8).One In a preferred embodiment, chimeric antibody is by the way that human kappa light chain constant region to connect to generate with mouse light chain variable region.Another In one preferred embodiment, chimeric antibody can be by the way that people's lambda light chain constant region to connect to generate with mouse light chain variable region.With In generate chimeric antibody preferred heavy chain constant region be IgG1, IgG3 and IgG4.For generate chimeric antibody other are preferred Heavy chain constant region is IgG2, IgA, IgD and IgM.
B) humanization
Antibody is mainly by being located at amino acid residue and target antigen in six heavy chains and light chain complementarity decision area (CDR) Interaction.Therefore, the amino acid sequence between single antibody in CDR than CDR outside sequence it is more various.Due to CDR sequences Row are responsible for the antibody-antigene interaction, specific naturally occurring it is therefore possible to express simulation by construction of expression vector The recombinant antibodies of the characteristic of antibody, the expression vector include the CDR sequence from the specific naturally occurring antibody, transplanting (such as Riechmann, L. etc. (1998) Nature is referred on to the Frame sequence from the different antibodies with different characteristics 332：323-327；Jones, P. etc. (1986) Nature 321：522-525 and Queen, C. etc. (1989) Proc.Natl.Acad.Sci.U.S.A.86：10029-10033).Such Frame sequence is available from comprising germ line antibody base Because of the public DNA database of sequence.These germline sequences will differ from ripe antibody gene sequences, because they do not include The variable gene completely assembled, the gene connect to be formed in B cell maturation by V (D) J.Germ line gene sequence Row will also be different from high-affinity secondary antibodies in a other places uniformly across variable region and compose (secondary repertoire Antibody sequence).For example, somatic mutation phase in the amino terminal portion of framework region 1 and the carboxy terminal half of framework region 4 To rare.In addition, many somatic mutations do not significantly change the binding characteristic of antibody.Therefore, resist to rebuild to have with original The intact recombinant antibody of the similar binding characteristic of body, it is not necessary that obtain the global DNA sequence of specific antibodies (refering to WO 99/ 45962).Partial heavy and sequence of light chain across CDR region are typically enough for the purpose.The partial sequence is used In determining that those system genitales are variable, and connect to recombinating the contributive section of antibody variable gene.Then germline sequence is used Fill up the part lacked in the variable region.Heavy chain and light chain leader sequence are cut during protein maturation, to final The characteristic of antibody is not contributed.It, can be by connection or PCR amplification by the cDNA sequence of clone and conjunction in order to add in the sequence lacked Into oligonucleotide combinatorial.Alternatively, complete variable region can be synthesized to one group of short overlapping oligonucleotide, and expanded by PCR Increasing is combined to produce complete variable region clone.This method has the advantages that, such as eliminates or comprising specific restricted Site or the specific codon of optimization.
The overlapping of synthetic oligonucleotide is designed using the heavy chain from hybridoma and the nucleotide sequence of light chain transcripts Group, to generate the synthesis V sequence identical with the amino acid encoding capacity of native sequences.The heavy chain and κ chain-orderings of synthesis can be with three Kind mode is different from native sequences：Repetition nucleotide base string is destroyed, in order to oligonucleotide synthesis and PCR amplification；According to Kozak rules be mixed with best translation initiation site (Kozak, 1991, J.Biol.Chem.266：19867-19870)；And Transformation obtains the HindIII sites of translation initiation site upstream.
For heavy chain and light chain variable region, the sequence of optimized coding strand and corresponding noncoding strand is in corresponding non-volume About 30-50 nucleotide is broken at the approximate midpoint of code oligonucleotides.It therefore, can be by the oligonucleotides for every chain It is assembled into the overlapping double-strand of the section across 150-400 nucleotide.Then the library is used as template and generates about 150-400 The pcr amplification product of nucleotide.It is two libraries usually by single variable region oligonucleotide component, each expands to generate two The PCR product of a overlapping.Then these overlapping products are combined by PCR amplification, to form complete variable region.Expand in PCR The overlapping fragments for including heavy chain or constant region of light chain may be needed in increasing, can easily be cloned into expression vector constructs with generating Segment.
Then by the promoter of the chimeric or humanized heavy chain and light chain variable region of reconstruction and clone, targeting sequencing, translation Homing sequence, constant region, 3 ' non-translated sequences, Polyadenylation sequences and transcription terminator combination, to form expression vector Construct.Then heavy chain and light chain expression constructs can be combined into single carrier, cotransfection, continuous transfection or transfect respectively into In host cell, then the host cell merges the host cell to form two kinds of chains of expression.It is described below to build people The plasmid of IgG κ expression vectors.The plasmid is built so that the V heavy chains and V κ light chain cDNA sequences of PCR amplification can be used for having rebuild Whole heavy chain and light chain minigenes (minigene).These plasmids can be used for expression human antibody or chimeric IgG1 κ or IgG4 κ Antibody.Similar plasmid can be built for expressing other heavy chain isotypes or expressing the antibody for including lambda light chain.
Therefore, in another aspect of the invention, generated in structure using the structure feature of the anti-CLD18 antibody of the present invention The relevant anti-CLD18 antibody of humanization, the humanized antibody retain the functional characteristic of at least one antibody of the present invention, such as combine CLD18.It more specifically, can be by recombination form by one or more CDR regions of mouse monoclonal antibody and known people's frame Area and CDR combinations, to generate the other anti-CLD18 antibody of the humanization through modified recombinant of the present invention.
It is combined with antigen-expressing cells
Standard binding assay can be used to be measured for the ability of antibody combination CLD18, such as the measure disclosed in embodiment (such as ELISA, western blot, immunofluorescence and flow cytometry).
The characterization combined to antibody
In order to purify anti-CLD18 antibody, selected hybridoma can be cultivated in 2 liters of centrifugal bottles, is resisted with Purified monoclonal Body.Alternatively, anti-CLD18 antibody can be generated in the bioreactor based on dialysis.It can with filtering supernatant and if necessary It is concentrated, carries out affinity chromatography with protein G-Sepharose or Protein A-agarose thereafter.Gel electrophoresis and efficient liquid can be passed through The IgG of phase chromatography inspection elution is to ensure purity.Buffer solution is commutative into PBS, and passes through OD280 using 1.43 extinction coefficient Carry out measured concentration.Monoclonal antibody can be divided into aliquot and be stored in -80 DEG C.
In order to determine whether selected anti-CLD18 monoclonal antibodies combine unique epitope, can use direct mutagenesis or Multipoint directional mutagenesis.
Determine isotype
In order to determine the isotype of purified antibody, a variety of commercially available reagent box (such as Zymed, Roche can be used Diagnostics isotype ELISA) is carried out.The hole of anti-mouse Ig coating microtiter plates can be used.After closing, in environment temperature Plate is made to be reacted with the isotype controls of monoclonal antibody or purifying under degree 2 hours.Then make hole and mouse IgG 1, IgG2a, IgG2b or IgG3, IgA or the reaction of Mouse IgM Specific peroxidase conjugated probe.After washing, with ABTS substrates (1mg/ Ml plate) is made to develop the color and OD is analyzed at 405-650.Alternatively, it can be resisted as described in manufacturer using IsoStrip mouse monoclonals Body isotype kit (Roche, catalog number (Cat.No.) 1493027).
Flow cytometry
In order to prove that there are anti-CLD18 antibody or proof monoclonal antibody combination expression in the serum of immune mouse The living cells of CLD18 can use flow cytometry.Can by the natural cell line expressed or expressed after CLD18 is transfected and Lack in negative control (being cultivated under standard growth conditions) and the doma supernatant of CLD18 expression or the PBS containing 1%FBS In a variety of concentration monoclonal antibody mixing, and can be incubated 30 minutes at 4 DEG C.After washing, phase can dyed with first antibody The anti-igg antibody that APC or Alexa647 is marked is made to be combined with combining the monoclonal antibody of CLD18 under conditions of.It can pass through Using the flow cytometry sample of FACS devices, wherein setting door to single living cell using light scattering and side scatter properties Control.In order to distinguish CLD18 monoclonal antibody specifics and non-specific binding thing in single measurement, cotransfection can be used Method.The cell for having transiently transfected the plasmid of coding CLD18 and fluorescent marker can be dyed as above-mentioned.Transfectional cell can be It is detected in the fluorescence channel different from antibody staining cell.Since most of transfectional cell expresses two kinds of transgenosis simultaneously, because This CLD18 monoclonal antibody specific is preferentially with expressing the cell combination of fluorescent marker, and non-specific antibody is with comparable ratio Rate is combined with non-transfected cells.As the supplement or replacement of Flow Cytometry Assay, replacing using fluorescence microscopy can be used It is measured for property.Above-mentioned staining cell can be pressed completely, and cell is checked by fluorescence microscopy.
If specific attached cell loses (for example, by cell detachment) and faces cytoproximal cell-cell contact, tightly Close connection albumen is usually internalized by.The cell surface expression of CLD18 can be adjusted by the following means：A) condition of culture is adjusted, such as It is cultivated by standard mode with higher cell density；Using mild disengaging condition (such as 2mM EDTA/PBS or Accutase enzymes), at room temperature handle and add endocytosis inhibitor (such as Sodium azide) CLD18 transcription or translation Activator and the cell for b) selecting and being cloned in cell surface maintenance high level CLD18, such as by using transfectional cell Antibiotic is selected, and by immune magnetic or FACS cell sortings and passes through limited dilution cloning.
Immunofluorescence microscopy
In order to prove that there are anti-CLD18 antibody or proof monoclonal antibody combination expression in the serum of immune mouse The living cells of CLD18 can be analyzed using immunofluorescence microscopy.For example, under standard growth conditions, supplemented with 10% tire Cow's serum (FCS), 2mM Pidolidones, 100IU/ml penicillin and 100 μ g/ml streptomysins DMEM/F12 culture mediums in room The spontaneous expression of culture or the cell line expressed after CLD18 is transfected and shortage CLD18 expression in slide (chamber slide) Negative control.Then cell methanol or paraformaldehyde are fixed or do not dealt with.Then make cell and needle at 25 DEG C To the monoclonal antibody reactive 30 minutes of CLD18.After washing, cell can be made to be marked with Alexa555 under the same conditions anti-small Mouse IgG secondary antibodies (Molecular Probes) are reacted.Then cell is checked by fluorescence microscopy.
When cell is fixed or paraformaldehyde is fixed and after carry out permeabilization with Triton X-100 with methanol, in observable cell Total CLD18 it is horizontal.It is fixed in cell in living cells and the paraformaldehyde of non-permeabilization, can check the surface positioning of CLD18.This Outside, it can altogether dye to analyze CLD18 to close-connected targeting by using close linkage flag such as ZO-1.Furthermore, it is possible to it examines Look into the effect that CLD18 is positioned in antibody combination and cell membrane.
Western blot
The reactivity of anti-CLD18IgG and CLD18 antigens can also be tested by western blot.In short, it can make The standby cell extract from CLD18 expression cells and appropriate negative control, and carry out lauryl sodium sulfate (SDS) poly- third Acrylamide gel electrophoresis.After electrophoresis, separated antigen is transferred to nitrocellulose filter, closes and is resisted with monoclonal to be tested Body is detected.Anti-mouse IgG peroxidase detection IgG can be used to combine, and developed the color with ECL substrates.
Immunohistochemistry
Can also the mode as known to technical staff anti-CLD18 mouse IgGs and CLD18 antigens are detected by immunohistochemistry Reactivity, such as the paraformaldehyde from non-cancer tissue or cancer tissue samples or acetone is used to fix freezing microtome section or paraffin packet Bury histotomy, the sample derived from during routine operation patient or derived from be vaccinated with autonomous expression (such as DAN-G, SNU-16 or KATO-III) or the cell line of expression (such as HEK293) CLD18 after transfection is small with xenograft tumours Mouse.For carry out immunostaining, antibody react with CLD18 can be inoculated with, thereafter according to manufacturer illustrate be inoculated be conjugated it is peppery The goat anti-mouse or goat anti-rabbit antibodies (DAKO) of root peroxidase.
Antibody phagocytosis in vitro and cell killing activity
Other than specifically binding CLD18, its mediate phagocytosis of CLD18 antibody tests and killing CLD18 can also be fought The ability of expression cell.The testing in vitro of monoclonal antibody activity model measurement will provide preliminary screening before in vivo.
The cytotoxicity (ADCC) of antibody dependent cellular mediation：
In short, pollution red blood cell can be then cracked by Ficoll Hypaque density centrifugations comes from health to purify The polymorphonuclear cell (polymorphonuclear cell, PMN) of donor, NK cells, monocyte, mononuclearcell or other Effector cell.Washed effector cell can be suspended from and inactivate human serum supplemented with 10% heat-inactivated fetal bovine serum or 5% heat In RPMI, and in a variety of effector cells and the ratio and use of target cell51The CLD18 expression target cell mixing of Cr labels.It alternatively, can To mark target cell with Fluorescence Increasing ligand (BATDA).Enhancing ligand and europium that dead cell is discharged can be measured by fluorimeter The high fluorescent chelate formed.Another substitute technology is using with luciferase transfection target cell.Then, the fluorescein of addition It can only be aoxidized by living cells.Then the anti-CLD18IgG of purifying can be added in a variety of concentration.Unrelated human IgG can be used as negative Control.Measuring can carry out 4 to 20 hours at 37 DEG C, depending on used effector cell's type.It can be by measuring on culture In clear liquid51The release of Cr or EuTDA chelates measure the cell cracking of sample there are situation.Alternatively, it is aoxidized by fluorescein Generated shine can be as the measurement of living cells.
Anti- CLD18 monoclonal antibodies can be tested with multiple combinations, to determine whether enhanced carefully with a variety of monoclonal antibodies Cellular lysate.
Complement-dependent cytotoxicity (CDC)：
The ability of a variety of known technologies its mediation of anti-CLD18 antibody tests CDC to monoclonal can be used.For example, can be with Mode obtains complement sera from blood known to technical staff.In order to measure the CDC of mAb activity, different methods can be used. For example, it can measure51Cr is discharged or can be excluded to measure to assess the raising of membrane permeability using propidium iodide (PI).Letter Yan Zhi, washable target cell simultaneously can be at room temperatures or 37 DEG C by 5 × 105A/ml and the mAb of a variety of concentration are incubated 10-30 minutes. Then serum or blood plasma can be added in final concentration of 20% (volume/volume), then incubated cell 20-30 minutes at 37 DEG C. All cells from each sample can be added in the PI solution in FACS pipes.Then can be led to immediately using FACSArray Overflow-type cytometry analyzes the mixture.In a kind of alternative measure, CDC can be measured to attached cell and lured It leads.In an embodiment of this measure, 24 hours with 3 × 10 before the assay4A cells/well is by cell inoculation into flat Tissue culture microtiter plate.Growth medium is removed, and is in triplicate incubated with cell and antibody within second day.To measure Background cracks and maximum cracking, and control cell is incubated respectively with growth medium or together with the growth medium containing 0.2% saponin(e It educates.Incubation at room temperature after twenty minutes, removes supernatant, and the human plasma of in DMEM 20% (volume/volume) or serum (are preheated to 37 DEG C) cell is added in, it is incubated again at 37 DEG C 20 minutes.All cells from each sample are added in into propidium iodide solution (10 μ G/ml in).Then supernatant is replaced with the PBS containing 2.5 μ g/ml propidium iodides, and Tecan is used after 520nm excitations Safire measures fluorescent emission at 600nm.Percent specific lysis is calculated as：The % Specific lytics=(fluorescent/back of the body Scape fluorescence)/(maximum cracking fluorescence-background fluorescence) × 100.
Cell Proliferation is inhibited by monoclonal antibody：
In order to test the ability of starting apoptosis, for example, can be by the anti-CLD18 antibody of monoclonal and CLD18 positive tumor cells It (such as SNU-16, DAN-G, KATO-III) or has transfected the tumour cell of CLD18 and has been incubated about 20 hours at 37 DEG C.It can harvest thin Born of the same parents are washed, and the annexin V with FITC or APC has been conjugated with annexin-V combination buffers (BD biosciences) (BD biosciences) is incubated 15 minutes in the dark.The PI that all cells from each sample can be added in FACS pipes is molten In liquid (10 μ g/ml in PBS), and assessed immediately by flow cytometry (as described above).Alternatively, commercially available reagent can be used Box detects the general cell inhibitory effect of monoclonal antibody.DELFIA cell proliferation reagents box (Perkin-Elmer, catalog number (Cat.No.) AD0200 it is) that the bromo- 2 '-BrdUs of 5- (BrdU) mix in the DNA building-up processes of proliferative cell based on measuring in microwell plate Heterotope immunoassays.The BrdU of incorporation is detected using the monoclonal antibody of europium label.To carry out antibody test, using solid Determine liquid and fix cell and denatured DNA.It washes away unbonded antibody and adds in DELFIA inducers, by europium ion from labelled antibody It dissociates in solution, they form high fluorescent chelate with the component of DELFIA inducers in the solution.(when using in the detection Between resolved fluorometric art) measure fluorescence it is proportional to the DNA synthesis of cell in each hole.
Preclinical study
Model (it such as can also be vaccinated with expression (such as DAN-G, SNU-16 or KATO-III) in vivo or express after transfection The immunodeficient mouse with xenograft tumours of the cell line of (such as HEK293) CLD18) in the list that is combined with CLD18 of test Clonal antibody, to determine the effect of the growth of tumour cell of its control expression CLD18.
In the tumour cell heterograft that will express CLD18 this can be used into after immunocompromised host mouse or other animals Invention antibody carries out In vivo study.Tumour cell can be injected thereafter, to measure the antibody to tumor free mouse administration of antibodies Pre- preventing tumor is formed or the effect of tumor-related symptoms.It can be to carrying the mouse administration of antibodies of tumour, to measure each antibody The effect of reducing tumour growth, transfer or tumor-related symptoms.Antibody can be applied and other substances such as cytostatic medicament, life Long factor inhibitors, Cell-Cycle Blockade agent, the application of angiogenesis inhibitor or other antibody are combined, to measure combination Synergistic effect and genotoxic potential.In order to analyze the antibody-mediated toxic side effect of the present invention, available antibodies or contrast agents inoculation are dynamic Object, and thoroughly research may be with the relevant symptom of CLD18 antibody therapies.The possible side effect of vivo applications CLD18 antibody is specific Include the toxicity of the tissue (including stomach and lung) to expressing CLD18.Identification CLD18's is anti-in people and other species (such as mouse) Body is particularly useful for predicting the potential side effect mediated in people using monoclonal CLD18 antibody.
Epitope mapping
Can exist " Epitope Mapping Protocols (Methods in Molecular such as Glenn E.Morris Biology) " in ISBN-089603-375-9 and Olwyn M.R.Westwood, Frank C.Hay in " Epitope Mapping：A Practical Approach " Practical Approach Series, the detailed description in 248 carry out this Invention antibody identifies the mapping of epitope.
I. bispecific/multispecific molecule of CLD18 is combined
In another embodiment of the present invention, can be derivatized for the antibody of CLD18 or with other functional moleculars (such as other peptide or protein matter, such as Fab ' segments) are connected, to generate the bispecific for combining multiple binding sites or target epitope Or multispecific molecule.For example, the antibody of the present invention can be with one or more other binding molecule (such as other antibody, peptide or knots Close analogue body) functionality connection (such as being connected by chemical coupling, genetic fusion, Non-covalent binding or other modes).
Therefore, the present invention includes bispecific and multispecific molecule, and it includes at least one the first combinations to CLD18 Specificity and the second binding specificity to the second target epitope.In one embodiment of the invention, the second target epitope Such as people Fc- γ RI (CD64) or people's Fc- α receptors (CD89) or it is T cell receptor for Fc receptors, such as CD3.Therefore, this hair Bright effector cell (such as monocyte, macrophage or polymorphonuclear cell including with expression Fc- γ R, Fc- α R or Fc- ε R And the target cell of expression the CLD18 bispecific and multispecific molecule that can react (PMN)).These bispecifics and Duo Te Opposite molecule can make the cell-targeting effector cell of expression CLD18, and can trigger the receptor-mediated effector cell's activity of Fc, such as The phagocytosis of CLD18 expression cells, cytotoxicity (ADCC), cytokine release or the super oxygen of antibody dependent cellular mediation The generation of anion.
Other than anti-Fc binding specificities and anti-CLD18 binding specificities, bispecific and polyspecific of the invention Molecule may also include third binding specificity.In one embodiment, third binding specificity is anti-enhancement factor (EF) portion Point, such as the molecule of the immune response for target cell is combined and thus improved with being related to the surface protein of cytotoxic activity.It is " anti- Enhancement factor part " can be antibody, functional antibody fragment or the ligand combined with given molecule (such as antigen or receptor), by This leads to enhance the effect that determinant is combined in Fc receptors or target cell antigen." anti-enhancement factor part " can be with Fc receptors or target Cellular antigens combine.It is combined in fact different from the first and second binding specificities alternatively, the anti-enhancement factor part can be combined The entity of body.For example, anti-enhancement factor part can combination cell poison T cell (such as by CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immunocytes that the immune response for target cell is caused to improve).
In one embodiment, bispecific of the invention and multispecific molecule are included as binding specificity extremely Few a kind of antibody, including such as Fab, Fab ', F (ab ')2, Fv or scFv.The antibody can also be light chain or heavy chain homodimer or Its any minimal segment of person, such as Fv or Ladner, US 4, the single-chain constructs described in 946,778.The antibody can also be Binding structural domain domain-immunoglobulin fusion proteins disclosed in US2003/0118592 and US 2003/0133939.
In one embodiment, bispecific of the invention and multi-specificity antibody include deposits on pairing effect cell surface Fc- γ R or Fc- α R binding specificity and another binding specificity to target cell antigen (such as CLD18).
In one embodiment, the binding specificity of Fc receptors is provided by monoclonal antibody, the combination of the antibody It is not blocked by immunoglobulin G (IgG).Terms used herein " IgG receptors " refer to eight γ chains on No. 1 chromosome Any one in gene.These gene codes totally 12 kinds of cross-films or soluble recepter isoform, are divided into 3 Fc- γ receptor classes： Fc- γ RI (CD64), Fc- γ RII (CD32) and Fc- γ RIII (CD16).In a preferred embodiment, the Fc- γ receptor behaviour high-affinity Fc- γ RI.
The generation of these preferred monoclonal antibodies and characterization are described in WO 88/00052 and US 4,954 by Fanger, 617.These antibody at the site different from receptor Fc- γ binding sites with Fc- γ RI, Fc- γ RII or Fc- γ RIII Epitope combines, therefore it combines and is not blocked significantly by the IgG of physiological level.The anti-Fc- γ RI of specificity for use in the present invention resist Body is mAb22, mAb32, mAb44, mAb62 and mAb197.In other embodiments, the anti-Fc- γ receptor antibodies Humanization form (H22) for monoclonal antibody 22.The generation of H22 antibody and characterization are described in Graziano, R.F. etc. (1995)J.Immunol.155(10)：4996-5002 and WO 94/10332.The cell for generating H22 antibody is lain in 1992 11 The moon 4 was preserved in American type culture collection with preserving number HA022CL1, and registration number is CRL 11177.
In other preferred embodiments, to the binding specificities of Fc receptors by combine people IgA receptors (such as Fc- α by Body (Fc- α RI (CD89))) antibody provide, combine preferably not by human immunoglobulin A (IgA) blocking.Term " IgA by Body " is intended to the gene outcome (Fc- α RI) for a α gene for including being located on No. 19 chromosomes.The known gene code 55 to The cross-film isoform of several alternative montages of 110kDa.Fc- α RI (CD89) are thin in monocyte/macrophage, acidophil granules Constitutive expression on born of the same parents and neutrophil cell, but do not expressed in non-effector cell group.Fc- α RI have IgA1 and IgA2 Medium affinity, this increase after the exposing cell factor such as G-CSF or GM-CSF (Morton, H.C. etc. (1996) Critical Reviews in Immunology 16：423-440).Describe except IgA ligand binding domains with Fc- Four kinds of Fc- α RI monoclonal antibody specifics that α RI are combined, are accredited as A3, A59, A62 and A77 (Monteiro, R.C. etc. (1992)J.Immunol.148：1764).
Fc- α RI and Fc- γ RI are for the preferred triggering receptor of the present invention, because their (1) are mainly expressed in immune effect It answers on cell, such as monocyte, PMN, macrophage and dendritic cells；(2) it is expressed at high levels that (such as 5000-100000/ is thin Born of the same parents)；(3) be cytotoxic activity (such as ADCC, phagocytosis) mediation person；(4) mediation is to targeting their antigen (including itself Antigen) enhancing antigen presentation.
In another embodiment, the bispecific molecule is by having the two of complementary functional activity Dan Ke of the present invention Grand antibody composition, such as mainly by inducing CDC, another antibody is mainly by apoptosis-induced come work come work for an antibody.
" effector cell's specific antibody " used herein refers to the antibody or functionality combined with the Fc receptors of effector cell Antibody fragment.For the present invention preferred antibody on the site for not combining endogenous immunoglobulin with effector cell's Fc receptor knots It closes.
Terms used herein " effector cell " refer to the identification of the effective stage for participating in immune response rather than immune response With the immunocyte of activation stage.Exemplary immune cells include the cell of marrow or lymphatic origin, and such as lymphocyte, (such as B is thin Born of the same parents and T cell, including cytolytic T cell (CTL), killing cell, natural killer cells, macrophage, monocyte, thermophilic Eosinophil, neutrophil cell, polymorphonuclear cell, granulocyte, mast cell and basophilic granulocyte.Some effector cells Specific Fc receptors are expressed, and perform specific immune function.In some preferred embodiments, effector cell can induce anti- Body dependent cellular cytotoxicity (ADCC), such as neutrophil cell can induce ADCC.For example, the monocyte of expression FcR, macrophage Cell participates in specific killing target cell and the other components for presenting antigens to immune system or the cell with presenting antigen With reference to.In other embodiments, effector cell can swallow target antigen, target cell or microorganism.Specific FcR is thin in effect Expression on born of the same parents can be adjusted by humoral factor such as cell factor.For instance, it has been found that interferon gamma (IFN-γ) up-regulation Fc- γ The expression of RI.The expression of this enhancing improves cytotoxic activity of the cell with Fc- γ RI to target.Effector cell can gulp down Bite or crack target antigen or target cell.
" target cell " should refer to undesired any cell in the subject (such as human or animal) that antibody of the present invention can target. In some preferred embodiments, the target cell is expression or the cell for being overexpressed CLD18.Express the cell one of CLD18 As include tumour cell.
Chemical technology can be used (to refer to such as D.M.Kranz (1981) Proc.Natl.Acad.Sci.USA 78： 5807), " polyoma (polydoma) " technology (referring to US 4,474,893) or recombinant DNA technology generate the bispecific of the present invention And multispecific molecule.
Specifically, methods known in the art can be used, pass through conjugating moiety binding specificity (such as anti-FcR and anti- CLD18 binding specificities) come prepare the present invention bispecific and multispecific molecule.For example, it can generate respectively double special Each binding specificity of property and multispecific molecule, is then conjugated each other.When binding specificity is protein or peptide, can incite somebody to action A variety of couplings or crosslinking agent are for covalently conjugated.The example of crosslinking agent include albumin A, carbodiimides, N- succinimidos- S- acetyl-thiacetate (SATA), 5,5 '-two thio two (2- nitrobenzoic acids) (DTNB), adjacent phenylenedimaleimide (oPDM), N- succinimidos -3- (2- pyridyldithios) propionate (SPDP) and sulfosuccinimide base -4- (N- horses Carrying out imide methyl) hexamethylene -1- carboxylates (sulfo group-SMCC) are (refering to such as Karpovsky (1984) J.Exp.Med.160：1686；Liu, MA etc. (1985) Proc.Natl.Acad.Sci.USA 82：8648).Other methods include Paulus (Behring Ins.Mitt. (1985) No.78,118-132)；(the Science (1985) 229 such as Brennan：81- And (the J.Immunol. (1987) 139 such as Glennie 83)：Method described in 2367-2375).Preferably conjugated agent for SATA and Sulfo group-SMCC is available from Pierce Chemical Co. (Rockford, IL).
When binding specificity is antibody, they can be bonded conjugated to occur by the sulfydryl of two heavy chain C-terminal hinge areas. In one particularly preferred embodiment, modification hinge region is to include odd number sulfhydryl residue, preferably 1 before conjugated.
Alternatively, two kinds of binding specificities can be encoded, and expressed simultaneously in same host cell in identical carrier Assembling.This method is mAb × mAb, mAb × Fab, Fab × F (ab ') in bispecific and multispecific molecule2Or ligand × It is particularly useful during Fab fusion proteins.The bispecific and multispecific molecule (such as bispecific molecule) of the present invention can be single Chain molecule, such as single chain bispecific antibody, the bispecific point for including a single-chain antibody and a combination determinant Son or the single chain bispecific molecule for including two basic change determinant.Bispecific and multispecific molecule can also be single-stranded Molecule can include at least two single chain molecules.The method for generating bispecific and multispecific molecule is described in such as US 5,260,203；US 5,455,030；US 4,881,175；US 5,132,405；US 5,091,513；US 5,476,786；US 5,013,653；US 5,258,498 and US 5,482,858.
Bispecific and multispecific molecule are combined with its specific target target can pass through enzyme linked immunosorbent assay (ELISA) (ELISA), radioimmunoassay (RIA), facs analysis, bioassay (such as growth inhibition) or western blot, which measure, comes It confirms.Each during these are measured generally all has the reagent of specific tape label (such as by using to purpose compound Antibody) detect the presence of specific purpose protein-antibody complexes.For example, FcR- antibody complexes can be used as identified simultaneously Enzyme-linked antibody or the antibody fragment of antibody-FcR complexes are specifically bound to detect.Alternatively, the compound can be used it is a variety of its Any one of his immunoassays detect.For example, radioactive label can be carried out to antibody, and for radioimmunoassay (RIA) it (refers to such as Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986).Radiation Property isotope can by such as using the method for gamma counter or scintillation counter come detect or by radioactive automatic developing come Detection.
II. immunoconjugates
In another aspect, the present invention is characterized in that treatment structure or medicament has been conjugated, (such as cytotoxin, drug (is such as exempted from Epidemic disease inhibitor) or radioactive isotope) anti-CLD18 antibody.Such conjugate is referred to herein as " immunoconjugates ".Packet It is known as " immunotoxin " containing one or more cytotoxic immunoconjugates.Cytotoxin or cytotoxic agent include having cell Evil particularly kills any medicament of cell.Example includes taxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecac It is alkali, mitomycin, Etoposide, Teniposide (tenoposide), vincristine, vincaleukoblastinum, colchicine, adriamycin, soft It is erythromycin, dihydroxy anthrax toxin diketone, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, general Shandong cacaine, totokaine, lidocaine, Propranolol and puromycin and the like or homologue.
The suitable therapeutic agent for being used to form immunoconjugates of the present invention includes but are not limited to antimetabolite (such as ammonia first is talked endlessly Purine, 6-MP, 6- thioguanines, cytarabine, fludarabine, 5 FU 5 fluorouracil dacarbazine), alkylating agent (such as mustargen, plug For sending Chlorambucil, melphalan, Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromo sweet dew Alcohol, streptozotocin, mitomycin C and suitable dichlorodiamine platinum (II) (DDP, cis-platinum), anthracycline (such as daunorubicin (daunorubicin is formerly referred to as daunomycin) and adriamycin), (such as actinomycin D (is formerly referred to as actinomyces to antibiotic Element), bleomycin, mithramycin and ammonia aspergillin (AMC) and antimitotic agent (such as vincristine and vincaleukoblastinum).One In a preferred embodiment, the therapeutic agent is cytotoxic agent or radiotoxicity agent.In another embodiment, it is described Therapeutic agent is immunosuppressor.In another embodiment, the therapeutic agent is GM-CSF.In a preferred embodiment In, the therapeutic agent is adriamycin, cis-platinum, bleomycin, sulfate, Carmustine, Chlorambucil, cyclophosphamide or castor-oil plant Toxin A.
The antibody of the present invention can also be conjugated with radioactive isotope (such as iodine 131, Y90 or indium 111), is controlled with generating Treat the radioactive cytotoxic drugs of CLD18 relevant diseases such as cancer.The antibody conjugates of the present invention can be used for changing what is given Biological response, and drug moiety should not be construed as limited to classical chemotherapeutant.For example, drug moiety can be expression The protein or polypeptide of required bioactivity.Such protein may include such as enzyme activity toxin or its active fragment, such as phase Think beans toxin, ricin A, Pseudomonas exotoxin or diphtheria toxin；Protein, such as tumor necrosis factor or interferon-γ； Or biological response changes agent, such as lymphokine, il-1 (" IL-1 "), proleulzin (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CSF ") or its His growth factor.
By these treatment structures and the technology of antibody conjugate to be known in the art, refering to such as Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", The editors such as Monoclonal Antibodies And Cancer Therapy, Reisfeld, 243-56 pages (Alan R.Liss, Inc.1985)；Hellstrom etc., " Antibodies For Drug Delivery ", in Controlled Drug The editors such as Delivery (second edition), Robinson, 623-53 pages (Marcel Dekker, Inc.1987)；Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy：A Review ", Monoclonal Antibodies ′84：The editors such as Biological And Clinical Applications, Pinchera, 475-506 pages (1985)；" Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ", Monoclonal Antibodies For Cancer The editors such as Detection And Therapy, Baldwin, 303-16 pages (Academic Press 1985) and Thorpe Deng, " The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates ", Immunol.Rev., 62：119-58(1982).
In another embodiment, antibody of the invention is attached to linker-chelator (as replaced smooth different shellfish not monoclonal antibody (tiuxetan)) on, the antibody is allowed to be conjugated with radioactive isotope.
III. pharmaceutical composition
In another aspect, the present invention provides the composition for including one or a combination set of antibody of the present invention, such as medicine group Close object.It can be according to routine techniques by described pharmaceutical composition and pharmaceutical acceptable carrier or diluent and any other known adjuvant Together with excipient, such as according to Remington：The Science and Practice of Pharmacy, the 19 editions, Gennaro is edited, the technology disclosed in Mack Publishing Co., Easton, PA, 1995.In an implementation In scheme, the composition includes the antibody of the present invention detached by a variety of (such as two or more) that different mechanisms act ons Combination, such as by mainly by induce a kind of antibody that CDC plays a role with mainly by it is apoptosis-induced play a role it is another One antibody combination.
The present invention pharmaceutical composition can also be applied in combination treatment, i.e., with other pharmaceutical agent combinations.For example, described group Closing therapy may include composition of the invention and at least one anti-inflammatory agent or at least one immunosuppressor.In an embodiment party In case, such therapeutic agent includes one or more anti-inflammatory agents, such as steroid drug or NSAID (non-steroidal antiinflammatory drugs, nonsteroidal anti-inflammatory drug).Preferred medicament include as aspirin and other salicylates, Cox-2 inhibitor such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAID classes such as brufen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), Diclofenac (Voltaren), Piroxicam (Feldene), Ketoprofen (Orudis), Diflunisal (Dolobid), Nabumetone (Relafen), Etodolac (Lodine), olsapozine (Daypro) and Indomethacin (Indocin)
In another embodiment, such therapeutic agent includes the medicament for causing regulatory T cells removing or functionally inactive, Such as cyclophosphamide, anti-CTLA 4 antibody, anti-IL2 or the anti IL2 receptor antibody of low dosage.
In another embodiment, such therapeutic agent includes one or more chemotherapeutants, such as taxol derives Object, Docetaxel (taxotere), gemcitabine, 5 FU 5 fluorouracil, adriamycin (Adriamycin), cis-platinum (Platinol), cyclophosphamide (Cytoxan, Procytox, Neosar).In another embodiment, antibody of the invention It can be applied with chemotherapeutic combination, the chemotherapeutant is preferably getting a cancer of the stomach, in the patient of the cancer of the esophagus, cancer of pancreas and lung cancer Show curative effect.
In another embodiment, antibody of the invention can be with radiotherapy and/or autologous peripheral blood stem cells or marrow Transplanting is administered in combination.
In another embodiment, antibody of the invention can be with applying selected from following one or more antibody combinations：It is anti- CD25 antibody, anti-EPCAM antibody, anti-EGFR, anti-Her2/neu and anti-CD 40 antibodies.
In another embodiment, antibody of the invention can be applied with anti-C3b (i) antibody combination, to enhance complement activation.
Any and all solvent of " pharmaceutical acceptable carrier " used herein including PHYSIOLOGICALLY COMPATIBLE, decentralized medium, coating, antibacterial Agent and antifungal agent, isotonic agent and absorption delaying agent etc..Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, stomach and intestine Outside, backbone or Epidermal administration (such as by injecting or being transfused).It, can be by reactive compound (i.e. antibody, Shuan Te depending on administration route The opposite sex and multispecific molecule) be wrapped in certain material, with protect compound from may make the inactivation acid and The effect of other natural endowments.
" officinal salt " refers to the desired bioactivity for retaining parent compound and does not lead to any undesired toxicity The salt for learning effect (refers to such as Berge, S.M., waits (1977) J.Pharm.Sci.66：1-19).
The example of such salt includes acid-addition salts and base addition salts.Acid-addition salts are included from non-toxic inorganic such as salt The salt of acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid etc. and from non-toxic organic acid such as aliphatic mono-carboxylic acids and The salt of dicarboxylic acids, benzene substitution alkanoic acid, hydroxy alkanoic acid, aromatic acid, fat and aromatic sulphonic acid etc..Base addition salts are included from alkaline earth The metal such as salt of sodium, potassium, magnesium, calcium etc. and from non-toxic organic amine such as N, N '-dibenzyl-ethylenediamin, N-METHYL-ALPHA-L-GLUCOSAMINE, chlorine The salt of procaine, choline, diethanol amine, ethylenediamine, procaine etc..
The composition of the present invention can be applied by a variety of methods known in the art.It will be appreciated by those skilled in the art that it gives Medicine approach and/or pattern will as expected result and change.Reactive compound can be with protecting the compound not discharge rapidly Carrier prepare together, such as controlled release preparation, including implantation material, transdermal patch and microcapsules delivery system.Can use can Biodegradable biocompatible polymer, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and poly- Lactic acid.The method for preparing these preparations is known to those skilled in the art.Refering to such as Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson are edited, Marcel Dekker, Inc., New York, 1978。
In order to by certain administration routes apply the compound of the present invention, it may be necessary to certain material package compound or It is co-administered with it, to prevent its inactivation.For example, compound can be applied in appropriate carrier such as liposome or diluent it is tested Person.Pharmaceutically acceptable diluent includes brine and aqueous buffer solution.Liposome includes W/O/W CGF emulsions and conventional liposome (Strejan etc. (1984) J.Neuroimmunol.7：27).
Pharmaceutical acceptable carrier includes aseptic aqueous solution or dispersant and for preparing aseptic parenteral solution or dispersion facing the used time The sterile powder of agent.These media and reagent are known in the art for the purposes of pharmaceutically active substance.In addition to any conventional Jie Other than matter or medicament are compatible with reactive compound, it is also contemplated that their purposes in pharmaceutical composition of the present invention.Complementarity Reactive compound can be also mixed in the composition.
Therapeutic composition generally has to be sterile, and stablizes producing under holding conditions.The composition can be prepared Into solution, microemulsion, liposome or other be suitable for high drug concentration ordered structure.Carrier can be containing such as water, ethyl alcohol, The solvent or decentralized medium of polyalcohol (such as glycerine, propylene glycol and liquid macrogol etc.) and its appropriate mixture.It can be with Such as by using coating (such as lecithin), by particle size desired by maintenance (for the dispersant in the case of) and by making Appropriate mobility is maintained with surfactant.In many cases, isotonic agent is preferably comprised in composition, such as sugared, more First alcohol such as mannitol, sorbierite or sodium chloride.It can be by including reagent (such as monostearate of delayed absorption in the composition Salt and gelatin) cause the extension of composition for injection to absorb.
Aseptic parenteral solution can be by by reactive compound and one kind in required mentioned component or being combined together with aequum It mixes appropriate solvent and then carries out sterile microfiltration to prepare.
Dispersant generally is prepared by the way that reactive compound is mixed sterile carrier, contains alkalinity point in the sterile carrier Dispersion media and other above-mentioned required ingredients.In the case of the sterile powder for being used to prepare aseptic parenteral solution, preferred preparation side Method is is dried in vacuo and freeze-drying (freeze-drying), the method obtain active constituent from the solution being sterile filtered before and add times The powder of what additional desired ingredient.
Dosage regimen is adjusted to provide best expectation response (such as therapeutic response).For example, single can be carried out It injects, can reduce at any time using several separated dosage or in proportion or improve according to the urgency level for the treatment of Dosage.Particularly advantageously parenteral composition is configured to dosage unit form, in order to apply and realize the uniform of dosage Property.Dosage unit form used herein refers to the physically separate unit for being suitable for subject single dose；Each unit In contain the reactive compound of predetermined amount associated with required pharmaceutical carrier, the predetermined amount is is computed generating desired treatment The amount of effect.The specification of dosage unit form of the present invention is determined by following factor, and directly depends on following factor：(a) it activates It closes the specific characteristic of object and is intended to the specific curative effect realized and (b) allotment for treating these work of sensibility situation in individual Property compound inherent limitation.
The example of pharmaceutically acceptable antioxidant includes：(1) water soluble antioxidant, for example, vitamin C, cysteine hydrochloride, Niter cake, sodium pyrosulfite, sodium sulfite etc.；(2) oil-soluble inhibitor, such as vitamine C palmitate, Butylated Hydroxyanisole (butylated hydroxyanisole, BHA), Butylated Hydroxytoluene (butylated hydroxytoluene, BHT), lecithin, Propylgallate, alpha tocopherol etc.；And (3) metal-chelator, such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorb Alcohol, tartaric acid, phosphoric acid etc..
For therapeutic combination, preparation of the invention includes being suitable for oral, intranasal, part (topical) (including mouth Chamber and sublingual), rectum, vagina and/or parenteral administration preparation.The preparation can be deposited routinely with dosage unit form , and can be prepared by method any known in pharmaceutical field.Single dosage form can be combined to produce with carrier material The amount of active constituent will change according to the object and specific mode of administration controlled.It can be combined to produce with carrier material single The amount of the active constituent of dosage form usually by be generate curative effect amount of composition.
Usually, in amounting to 100%, the range of this amount will be 0.01% to about 99% active constituent, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%.
The vaginal suppository containing suitable carrier known in the art, health are further included suitable for the invention formulation of vagina administration Sliver, cream, gelling agent, Past, foaming agent or spray.For part or the dosage form of the transdermal administration present composition Including pulvis, spray, ointment, Past, cream, lotion, gelling agent, solution, patch and inhalant.Active ingredient Object can aseptically with pharmaceutical acceptable carrier and any preservative, buffer or the propellants that may need.
Terms used herein " parenteral administration " and " parenteral administration " refer to the administration mould of non-enteral and non local administration Formula typically by injection, includes but are not limited to that intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, socket of the eye is interior, intracardiac, corium In interior, peritonaeum, under transtracheal, subcutaneous, epidermis, under intra-articular, capsule, under arachnoid, intraspinal tube, Epidural cavity, breastbone inner injection and Infusion.
The example of appropriate aqueous and non-aqueous carrier available for pharmaceutical composition of the present invention includes water, ethyl alcohol, polyalcohol (such as glycerine, propylene glycol, polyethylene glycol) and its appropriate mixture, vegetable oil (such as olive oil) and injection organic ester (such as ethyl oleate).It can be by such as using coating material (such as lecithin), by maintaining desired particle size (for dispersant Situation) and maintain by using surfactant appropriate mobility.
These compositions can also contain adjuvant, such as preservative, wetting agent, emulsifier and dispersant.By disinfecting action and By can prevent comprising a variety of antiseptics and antifungal agent (such as p-hydroxybenzoate, anesin, phenol, sorbic acid) Only there are microorganisms.It can also expect in the composition comprising isotonic agent, such as sugar, sodium chloride.Furthermore, it is possible to by comprising delaying The medicament (such as aluminum monostearate and gelatin) of absorption come realize the extension of injectable drug form absorb.
In one embodiment, by hypodermic injection using the monoclonal antibody of the present invention of crystal form, refering to Yang Deng (2003) PNAS, 100 (12)：6934-6939.When the present invention composition as medicament administration in humans and animals when, they Can be administered alone or be administered as pharmaceutical composition, described pharmaceutical composition include such as 0.01% to 99.5% (more preferable 0.1 to 90%) combination of active constituent and pharmaceutical acceptable carrier.
No matter any administration route is selected, it can the present composition that suitably hydrated form uses and/or the present invention Pharmaceutical composition is configured to pharmaceutically acceptable dosage form by conventional method well known by persons skilled in the art.
The definite dosage level of active constituent in pharmaceutical composition of the present invention can be changed, so as to specific patient, group It closes object and ideal therapeutic response and the active principle nontoxic to patient is effectively realized in mode of administration acquisition.Selected dose Amount level will depend on a variety of pharmacokinetics factors, and activity, administration route including the specifically used present composition are given Medicine time, the discharge rate of specifically used compound, duration for the treatment of, its used with specifically used combination of compositions His drug, compound and/or material, by control age of patient, gender, weight, illness, general health and medical history before and Similar factor known to medical domain.
This field gengral practitioner or animal doctor can be readily determined the effective quantity of required pharmaceutical composition and output prescription.Example Such as, doctor or animal doctor can give the present invention used in pharmaceutical composition since less than the level for obtaining dosage needed for Preferred effects The dosage of compound, and dosage is gradually increased until realizing ideal curative effect.In general, the appropriate present composition is every Daily dose by be effective lowest dose level for generating curative effect chemical combination object amount.Such effective dose generally will depend on it is above-mentioned because Element.It is preferred that in intravenous, intramuscular, peritonaeum or subcutaneous administration, preferably it is applied near target area.When necessary, therapeutic combination Daily dose can with 2,3,4,5,6 or more low dose separate administration in one day is spaced in appropriate, optionally with Unit dosage form is applied.While it may be possible to the compounds of this invention is administered alone, but preferably using compound as pharmaceutical preparation (group Close object) application.
In one embodiment, antibody of the invention can be applied by being transfused, and preferably (it is small such as be longer than 24 in long-time When) in continuous infusion at a slow speed, so as to reduce toxic side effects.Using the time 2 to 24 hours (such as 2 to 12 hours) can be passed through Middle continuous infusion carries out.Such scheme can be repeated one or more times if necessary, such as the weight behind 6 months or 12 months It is multiple.Can Dan Ke in cycle be measured by using the anti-idiotype for targeting anti-CLD18 antibody in the biological sample after application The amount of grand anti-CLD18 antibody determines or adjusts dosage.
In another embodiment, pass through maintenance therapy (such as in 6 months or longer time once a week) administration of antibodies.
In another embodiment, the antibody of the present invention can be applied by including following scheme：Infusion needle is to CLD18's Antibody is primary, and infusion is conjugated with the radioisotopic antibody for CLD18 thereafter.The program can weigh after such as 7 to 9 days It is multiple to carry out.
Medical Devices known in the art can be used to apply for therapeutic combination.For example, in a preferred embodiment, Needlefree injection equipment can be used to apply for the therapeutic combination of the present invention, such as US 5,399,163, US 5,383,851, US 5,312,335th, the equipment disclosed in US 5,064,413, US 4,941,880, US 4,790,824 or US 4,596,556.It can Include those described in documents below for the well-known implants of the present invention and the example of module：US 4,487,603, it is public It has opened to can plant micro- infusion pump with controllable rate dispersion drug；US 4,486,194, it discloses for passing through skin Using the therapeutic equipment of drug；US 4,447,233, it discloses for the medical infusion of precise hydrodynamics rate-delivery drug Pump；US 4,447,224 can plant infusion device it discloses the variable flow rate for continuous drug delivery；US 4,439, 196, it discloses the penetrating pharmaceutical delivery systems with multi-cavity compartment；And US 4,475,196, disclosed permeability Drug delivery system.
Many other such implantation materials, delivery system and module are known to the skilled in the art.In certain implementations In scheme, antibody of the invention can be configured to ensure correctly distribution in vivo.For example, blood-brain barrier (blood-brain Barrier, BBB) eliminate many high-hydrophilic compounds.In order to ensure the therapeutic compounds of the present invention passes through BBB (necessary When), it can be for example prepared in liposome.Produce liposome method refer to as US 4,522,811, US 5,374,548, With US 5,399,331.Liposome may include one or more selective transports to the part in specific cells or organ, thus The delivering for enhancing targeted drug (refers to such as V.V.Ranade (1989) J.Clin.Pharmacol.29：685).Exemplary target to Part include folate/ester or biotin (US 5,416,016 for referring to such as Low), mannoside (Umezawa etc., (1988)Biochem.Biophys.Res.Commun.153：1038), antibody (P.G.Bloeman etc. (1995) FEBS Lett.357：140；M.Owais etc. (1995) Antimicrob.Agents Chemother.39：And surfactant egg 180) White A receptors (Briscoe etc. (1995) Am.J.Physiol.1233：134).
In one embodiment of the invention, therapeutic compounds of the invention is prepared in liposome.It is more excellent at one In the embodiment of choosing, the liposome includes targeting moiety.In a most preferred embodiment, the treatment in liposome Compound is delivered to the position of target site (such as tumor locus) nearby by injecting.The mobility of the composition must reach easy In the degree of injection.It must stablize, and must prevent microorganism (such as bacterium and fungi) producing under holding conditions It is preserved under conditions of contamination.
In another embodiment, antibody of the invention, which can be configured to prevent or reduce it, passes through the transhipment of placenta.This can It is realized by means known in the art, such as by the PEGylated of antibody or by using 2 ' segments of F (ab).It reference may also be made to " Cunningham-Rundles C, Zhou Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates.Resistance to enzymatic Degradation.J.Immunol.Methods, 152：177-190 and " Landor M. (1995) Maternal-fetal Transfer of immunoglobulins, Ann.Allergy Asthma Immunol.74：279-283.
It can be weighed for " treatment effective dose " of oncotherapy by target tumor response, the response can be Fully or partially.Complete response (complete response, CR) is defined as the clinic, radioactivity or other signs of no disease. Part response (partial response, PR) is since the reduction of tumor size being added up to be higher than 50%.Process (progression) median time is a measurement for characterizing objective tumor response durability.
It can also be weighed for " treatment effective dose " of oncotherapy by the ability of its stable disease process.Compound Inhibiting the ability of cancer can be evaluated in the animal model system for predicting its effect in human tumour.Alternatively, compound This characteristic can check that the compound inhibits cell growth or the ability of apoptosis by using external test known to technical staff To evaluate.The therapeutic compounds of therapeutically effective amount can reduce tumor size or improve the symptom of subject.People in the art Member can determine such measure based on the following factors：Such as the weight of subject, the severity of subject's symptom and selection Concrete composition or administration route.
The composition must it is sterile, and its mobility reach can delivery by injection degree.In addition to water, it carries Body can also be the saline solution, ethyl alcohol, polyalcohol (such as glycerine, propylene glycol and liquid macrogol) of isotonic buffer and its fit When mixture.It can be for example by using coating (such as lecithin), by maintaining ideal particle size (for dispersant Situation) and maintain by using surfactant appropriate mobility.In many cases it is preferred to include in the composition etc. Penetration enhancer, such as sugar, polyalcohol such as mannitol or sorbierite and sodium chloride.It can be by including the medicine of delayed absorption in the composition The Long-term absorption of composition for injection is realized in agent (such as aluminum monostearate or gelatin).
When reactive compound for example it is above-mentioned carried out appropriate protection when, the mixture can oral administration, it is such as dilute with inertia It releases agent or absorbent edible carrier is administered together.
IV. purposes and method of the invention
Antibody of the present invention (including immunoconjugates described herein, bispecific/multispecific molecule, composition and Other derivatives) there are a variety for the treatment of effectiveness, including treatment and the relevant illness of CLD18 expression cells.For example, the antibody can be applied For the cell (such as external or in vitro) or people experimenter (as in vivo) in culture, to treat or prevent various diseases, such as this The text illness.Terms used herein " subject " are intended to include response in the people and non-human animal of the antibody for CLD18. Preferred subject, which includes suffering from, (to be particularly characterized as that CLD18 expresses mould compared with normal cell by killing diseased cells The cell that formula changes) come the people patient of illness treating or improve.
Functional characteristic that the effect for the treatment of described herein is preferably killed by antibody directed cellular of the present invention realizes, example Such as split by what the cracking of inducing complement dependent cellular cytotoxicity (CDC) mediation, antibody-dependent cytotoxicity (ADCC) mediated Solution, apoptosis, homotypic adhesion and/or the killing of phagocytosis mediation, the preferably cracking of induction CDC mediations and/or ADCC mediations Crack the killing of mediation.
For example, in one embodiment, antibody of the invention can be used for the subject that oncogenicity illness is suffered from treatment, such as Suffer from the subject for the disease (including such as gastric cancer) for being characterized as the tumour cell for existing expression CLD18.The cause that can be treated and/or prevent The example of knurl disease include it is all expression CLD18 cancers and tumor entity, including gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, Oophoroma, breast cancer, colorectal cancer, liver cancer, gallbladder cancer and head and neck cancer.These cancers can be early stage, mid-term or late period, example Such as metastatic carcinoma.
Pharmaceutical composition and therapy of the present invention can be additionally used in immune or vaccine inoculation, to prevent disease described herein Disease.
In another embodiment, antibody of the invention can be used for detection CLD18 or specific CLD18 forms level or The level of person's detection cell containing CLD18 in its film surface, then can be by horizontal and certain diseases or the disease symptoms (as described above) is associated.Alternatively, above-mentioned antibody can be used for eliminating the function of CLD18 expression cells or interact therewith, thus By these cells as the important mediator of these diseases.This can be by making sample and control sample and anti-CLD18 antibody It contacts to realize under conditions of antibody is allowed to form compound with CLD18.To sample and control sample (i.e. reference sample) The alloy formed between antibody and CLD18 is detected, and is compared.
Can first in vitro to the antibody test of the present invention its with treatment or diagnostic uses relevant combination activity.For example, Flow cytometry as described herein can be used to measure the antibody.
It (is expressed in the effector cell's activity for triggering at least one effector mediation including inhibiting in addition, antibody can be measured The cell growth of CLD18 and/or the killing cell) activity.For example, the energy of antibody triggering CDC and/or apoptosis can be measured Power.The operation for measuring CDC, homotypic adhesion, molecular cluster or apoptosis is as described herein.
The antibody of the present invention can be used for causing one or more following biological activities in vivo or in vitro：Inhibit expression The cell growth of CLD18 and/or differentiation；The cell of killing expression CLD18；The mediation expression CLD18 in the presence of effector cell The phagocytosis of cell or ADCC；The CDC of the cell of mediation expression CLD18 in the presence of complement；The cell of mediation expression CLD18 Apoptosis；Induce homotypic adhesion；And/or induction is migrated after CLD18 is combined to Lipid Rafts (lipid raft).
In a specific embodiment, the antibody is a variety of for treating, preventing or diagnosing in vivo or in vitro CLD18 relevant diseases.The example of CLD18 relevant diseases including cancer etc., such as gastric cancer, cancer of pancreas, the cancer of the esophagus, lung cancer and above The cancer listed.
CLD18A2 is also expressed in the normal gastric cells of differentiation.Possibly through parallel application stomach protectiveness drug as resisted Sour agent or stomach proton pump inhibitor such as Omeprazole or related drugs come reduce or avoid antibody by kill these cells and The clinical side effects of induction.
Antibody compositions of the present invention are in vivo and external suitable administration route is known in the art, and can be by common Technical staff is selected.
As described above, anti-CLD18 antibody of the invention can (such as cytotoxic agent be put with one or more other therapeutic agents Penetrating property toxic agent, antiangiogenic agent or/and immunosuppressor) it co-administers, it should for the immune of antibody of the present invention to reduce induction It answers.Antibody can connect (as immune complex) with the medicament or can be with the medicament separate administration.For latter Situation (separate administration), antibody can before, after or at the same time be applied in medicament or can be (such as anti-with other known therapies Cancer therapy is such as radiated) it co-administers.Such therapeutic agent includes antitumor agent listed above etc..The anti-CLD18 antibody of the present invention with The co-application of chemotherapeutant provides two kinds of anticancer agents, they are played a role by different mechanisms, are obtained to tumour cell Cellulotoxic effect.Such co-application can solve to lead due to generating to change to the resistance or tumor-cell antigen of drug The problem of cause, described problem may be such that they do not react with the antibody.
In another embodiment of the present invention, to the subject of administration of antibodies additionally using antiangiogenic agent into Row treatment, the antiangiogenic agent include the antibody of targeting VEGF or VEGFR and inhibit the one or more of angiogenesis Compound.Pretreatment is carried out with these drugs or parallel can improve infiltration of the antibody in bulk tumour using these drugs.
In another embodiment of the present invention, the subject of administration of antibodies is additionally used and inhibits growth factor receptor The compound of body is treated, and the compound includes the monoclonal antibody combined with EGFR receptors and inhibits EGFR, Her1 Or the compound of signal that Her2/neu receptors are originated.
Can also use target specificity effector cell (such as with composition (such as antibody, polyspecific and bispecific point Son) connection effector cell) as therapeutic agent.Effector cell for targeting can be human leukocytes, as macrophage, it is thermophilic in Property granulocyte or monocyte.Other cells include eosinophil, natural killer cells and other with IgG or IgA by The cell of body.When necessary, effector cell is available from subject to be treated.Target specificity effector cell can connect in physiology It is applied in by solution as cell suspension.The cell number applied can be 108To 109The order of magnitude, but can be according to therapeutic purposes And change.In general, the amount will be enough to obtain the positioning at target cell (tumour cell as expressed CLD18), and pass through Such as cell killing is realized in phagocytosis.Administration route can also change.
It can combine progress with for removing the other technologies of target cell using the therapy of target specificity effector cell.Example Such as, the effector cell using the antitumor therapy of the present composition and/or with these compositions can join with chemotherapy It closes and uses.In addition it is possible to use two different cellulotoxic effect sub-groups are oriented to tumor cell rejection by combined immunization therapy. For example, the anti-CLD18 antibody being connect with anti-Fc-RI or AntiCD3 McAb can be used in combination with IgG or IgA receptor specific binding agents.
The bispecific and multispecific molecule of the present invention can be additionally used in Fc- γ R or Fc- the α R water on mediating effect+6 cell It is flat, such as carried out by covering with the receptor on scavenger-cell surface.The mixture of anti-Fc receptors can also be used for this purpose.
Can also be used in the presence of complement with complement-binding site (such as from IgG1,2 or 3 or IgM with complement knot The part of conjunction) the present composition (such as antibody, bispecific and multispecific molecule and immunoconjugates).In an implementation In scheme, can by add in complement or serum containing complement to use bonding agent of the present invention and appropriate effector cell to comprising The vitro treatment that the cell mass of target cell carries out is supplemented.Being coated with the phagocytosis of the target cell of bonding agent of the present invention can lead to It crosses conjugated complement albumen and is improved.In another embodiment, the target cell for being coated with the present composition can also be by complement Cracking.In another embodiment, composition of the invention not complement activation.
The composition of the present invention can also be applied together with complement.Therefore, comprising antibody, polyspecific or bispecific molecule And the composition of serum or complement is within the scope of the invention.The advantages of these compositions, is that complement is located at antibody, mostly spy Near the opposite sex or bispecific molecule.
Alternatively, antibody, polyspecific or the bispecific molecule and complement or serum of the present invention can separate administrations.This hair The combination of bright composition and target cell causes CLD18 antigen-antibody complexes to migrate into the Lipid Rafts of cell membrane.Such migration The high density antigen-antibody complex of CDC can effectively be activated and/or enhance by generating.
Kit comprising the present composition (such as antibody and immunoconjugates) and its operation instruction is also the present invention In the range of.The kit can also contain one or more other reagents, such as immunosuppressor, cytotoxic agent or radioactive poison Agent or one or more others antibody of the present invention (as having the antibody of complement activity).
Therefore, in addition the patient treated using antibody of the present invention can also be applied (before application antibody of the present invention, together When or later) enhancing or promote the other therapeutic agents of antibody curative effect of the present invention, such as cytotoxic agent or radioactivity toxic agent.
In other embodiments, can additionally by such as with cytokine therapy subject come using other medicaments come The subject is treated, wherein the medicament adjusts the expression or activity of (as enhanced or inhibiting) Fc- γ or Fc- α receptors.Preferably Cell factor includes granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-GSF), does Disturb element-γ (IFN-γ) and tumor necrosis factor (TNF).For improve antibody described herein and pharmaceutical composition curative effect other Important medicament is beta glucan, it is the homopolysaccharide of branch's glucose residue, is generated by various plants and microorganism, such as carefully Bacterium, algae, fungi, yeast and cereal.Biogenic beta glucan segment also can be used.Preferably, the beta glucan is β The polymer of (1,3) glucose, wherein at least some main chain glucose units (the main chain glucose unit of such as 3-6%), which have, to be divided Branch, such as β-(1,6) branch.
In a specific embodiment, the present invention is provided to detect the presence or measurement of CLD18 antigens in sample The method of CLD18 amount of antigen, including making sample and control sample in the item for allowing antibody or part thereof and CLD18 formation compounds The antibody of contact specific binding CLD18 under part.Then the formation of compound is detected, wherein sample is compound compared with control sample There are CLD18 antigens in the difference instruction sample that object is formed.
In another embodiment, the present invention is provided to detect CLD18 expression cells in vivo or in vitro or it is determined The method of amount.
Subject's application is conjugated the method includes (i) present composition of detection label；(ii) make this tested Person contacts the means of the detection detection label, to identify the position containing CLD18 expression cells.
Particularly, method as described above can be used for diagnosis CLD18 relevant diseases and/or positioning CLD18 relevant diseases, such as cancer Disease.Preferably, in sample CLD18 (preferably CLD18-A2) amount of the amount higher than CLD18 (preferably CLD18-A2) in control sample Indicate that there are CLD18 relevant diseases in the source subject (particularly people) of the sample.
In another embodiment, immunoconjugates of the invention can be used for compound (such as therapeutic agent, label, cell Toxic agent, radioactivity toxic agent, immunosuppressor etc.) targeting surface expression CLD18 cell, this is by the way that these compounds are resisted with this Body connects to realize.Therefore, the present invention also provides the in vitro or cell in vitro of localization and expression CLD18 (as the tumour in recycling is thin Born of the same parents) method.
The present invention is further illustrated by the following examples, the range that embodiment should not be construed as limiting the invention.
Embodiment
1. generate the mouse antibodies for CLD18
A. it is immunized：
With encoding human CLD18 segments (SEQ ID NO：15th, 16,17,18) carrier for expression of eukaryon be immunized Balb/c or C57/BL6 mouse.In the presence of adjuvant (such as CpG), on day 1 with the 10th day by 50 μ g or 25 μ g plasmid DNA injections into four-head Flesh (intramuscular, i.m.), for generate the 1st group of monoclonal antibody either on day 1 with the 9th day, the 1st day and the 11st day or the 1st My god, the 16th day and the 36th day carry out, for generating the 2nd group of antibody (details is shown in Table 1b).It intramuscular injection CpG and only transfects CLD18A2(SEQ ID NO：1) or the cell of cotransfection CLD18A2 and mouse soluble CD40L coding RNAs, intramuscular or peritonaeum Interior injection PEI-Man.According to specific immunization protocol used, supervised between the 16th day and the 43rd day by immunofluorescence microscopy Survey mice serum in for people CLD18 antibody there are situations.Using HEK293 raji cell assay Raji immunofluorescences, the cell wink When transfection have coding comprising people CLD18A2 (SEQ ID NO：1st, 2) and the nucleic acid of the fusion constructs of fluorescent reporter protein.Pass through Injection 5 × 10 in peritonaeum7It is a or 1 × 108It is a to have transiently transfected encoding human CLD18A2 (SEQ ID NO：1st, 2) nucleic acid HEK293 cells are strengthened the mouse with detectable immune response (Fig. 1) in 3 days before spleen is extractd, to generate the 1st group Monoclonal antibody is either strengthened mouse or 4 days before spleen is extractd, 3 for 3 days, 3 days and 2 days before spleen enucleation It and mouse was strengthened in 2 days, to generate the 2nd group of monoclonal antibody (details is shown in Table 1b).It is used in table 1a Immunization protocol is used for each monoclonal antibody.
Table 1a：For generating the immunization protocol of monoclonal antibody
*Specific immunization protocol is shown in Table 1b
B. the hybridoma for the human monoclonal antibodies for producing anti-CLD18 is generated：
Mouse boosting cell is detached based on standard scheme, and is merged using PEG with murine myeloma cell.Then it uses The HEK293 cells of the nucleic acid of encoding human CLD18 have been transfected, the production of CLD18 specific immunoglobulins is directed to by facs analysis The hybridoma that raw screening obtains.Using 50%PEG (Roche Diagnostics, CRL 738641), with 2: 1 ratio in the future From the splenic lymphocytes single cell suspension of immune mouse and P3X63Ag8U.1 nonsecreting mouse myeloma cells (ATCC, CRL 1597) it merges.By cell with about 3 × 104A/hole is taped against in flat-bottom microtiter plates, thereafter containing 10% fetal calf serum, 2% hybridoma fusion and clone additive (HFCS, Roche Diagnostics, CRL 1 363 735) plus 10mM HEPES, 2 are incubated in the Selective agar medium of 0.055mM2- mercaptoethanols, 50 μ g/ml gentamicins and 1 × HAT (Sigma, CRL H0262) Week.After 10 to 14 days, anti-CLD18 monoclonal antibodies are screened to individual cells by flow cytometry.By the hybridization of secretory antibody Knurl bed board again, is screened again, is still then subcloned for positive anti-CLD18 monoclonal antibodies by limiting dilution.Then body The stable subclone of outer culture, is characterized with generating a small amount of antibody in culture medium for tissue culture.It is selected from each hybridoma Select the clone of at least one reservation parental cell reactive (being measured by FACS).9 solencyte libraries are generated, and protect to each clone There are in liquid nitrogen.
C. the monoclonal antibody combined with CLD18 is selected：
In order to determine the isotype of antibody, isotype ELISA is carried out.Using mouse monoAB ID kits (Zymed, CRL 90-6550) or IsoStrip mouse monoclonal antibody isotypes kit (Roche, catalog number (Cat.No.) 1493027) measure The Ig subclass of identified CLD18 reactivity monoclonal antibody.It produces and is defined as the 1st group of 19 hybridoma cell lines, 6 are come From with CLD18A2-loopD3 (SEQ ID NO：17th, 18) fusions of immune C57/BL6 mouse cell, 18 come from CLD18A2-loop1(SEQ ID NO：15th, 16) fusions for the Balb/c mouse cells being immunized, they express following antibody：
24H5,26B5,26D12,28D10,37G11,37H8,38G5,38H3,39F11,41C6,42E12,43A11, 44E10,47D12,61C2,75B8,85A3,9E8,19B9
24H5：Mouse monoclonal IgG2b, κ antibody, 182-D758-034
26B5：Mouse monoclonal IgG2a, κ antibody, 182-D758-035, DSM ACC2745
26D12：Mouse monoclonal IgG3, κ antibody, 182-D758-036, DSM ACC2746
28D10：Mouse monoclonal IgG3, κ antibody, 182-D758-040, DSM ACC2747
37G11：Mouse monoclonal IgG2a, κ antibody, 182-D1106-055, DSM ACC2737
37H8：Mouse monoclonal IgG3, κ antibody, 182-D1106-056, DSM ACC2738
38G5：Mouse monoclonal IgG3, κ antibody, 182-D1106-057, DSM ACC2739
38H3：Mouse monoclonal IgG3, κ antibody, 182-D1106-058, DSM ACC2740
39F11：Mouse monoclonal IgG3, κ antibody, 182-D1106-059, DSM ACC2741
41C6：Mouse monoclonal IgG2a, κ antibody, 182-D1106-060
42E12：Mouse monoclonal IgG2a, κ antibody, 182-D1106-061, DSM ACC2748
43A11：Mouse monoclonal IgG2a, κ antibody, 182-D1106-062, DSM ACC2742
44E10：Mouse monoclonal IgG3, κ antibody, 182-D1106-063
47D12：Mouse monoclonal IgG3, κ antibody, 182-D1106-064
61C2：Mouse monoclonal IgG2b, κ antibody, 182-D1106-067, DSM ACC2743
75B8：Mouse monoclonal IgM, κ antibody, 182-D756-001
85A3：Mouse monoclonal IgM, κ antibody, 182-D756-002
9E8：Mouse monoclonal IgM, κ antibody, 182-D758-011
19B9：Mouse monoclonal IgM, κ antibody, 182-D758-024
It produces and is defined as the 2nd group of 5 hybridoma cell lines, 1 comes from CLD18A2-loopD3 (SEQ ID NO： 17th, 18) and CLD18A2-loopD1 (SEQ ID NO：15th, 16) fusions of immune Balb/c mouse cell, 4 come from CLD18A2-loopD1(SEQ ID NO：15th, 16) fusions for the Balb/c mouse cells being immunized, they express following antibody：
45C1,125E1,163E12,166E2,175D10
45C1：Mouse monoclonal IgG2a, κ antibody, 182-D758-187
125E1：Mouse monoclonal IgG2a, κ antibody, 182-D1106-279, DSM ACC2808
163E12：Mouse monoclonal IgG3, κ antibody, 182-D1106-294, DSM ACC2809
166E2：Mouse monoclonal IgG3, κ antibody, 182-D1106-308
175D10：Mouse monoclonal IgG1, κ antibody, 182-D1106-362, DSM ACC2810
2. generate monoclonal antibody
It generates and purifies the monoclonal antibody that there is reactivity to CLD18：
It is characterized to generate the antibody of mg amounts for function, with 2 × 106Hybridoma is inoculated into base by a cell/ml In the bioreactor (CELLine CL1000, Integra, Chur, CH) of dialysis.It harvests weekly once containing the upper of antibody Clear liquid.It is carried out using MelonGel (Pierce, Rockford, USA) purified mouse monoclonal antibodies and by ammonium sulfate precipitation Concentration is purified using FPLC by albumin A.Antibody concentration and purity are determined, and pass through 12 by BCA measure Sodium alkyl sulfate gel electrophoresis and coomassie dye to check purity.
3. the binding characteristic of monoclonal antibody
The quality control of body is transfected in a.WB, IF：
In order to generate the cell of expression CLD18A2, with coding CLD18A2 (SEQ ID NO：1st, 2) or CLD18A2-myc (SEQ ID NO：3rd, 4) nucleic acid transfection HEK293 or Chinese hamster ovary celI.
With CLDN18A2-myc (SEQ ID NO：3rd, 4) transfected HEK 293 or without transfection.24 is small after transfection When harvest cell, crack and carry out sodium dodecyl sulfate gel electrophoresis.Trace is carried out to gel and is contaminated with the anti-myc antibody of mouse Color.After being incubated with the anti-mouse antibody of peroxidase labelling, trace is made to develop the color, and be imaged using LAS-3000 with ECL reagents Instrument (Fuji) develops.The band with CLD18-myc estimated molecular weights is only observed in transfectional cell, and in negative control then Do not have (Fig. 2).
With CLD18A2 (SEQ ID NO：1,2) it transfection CHO cell and is cultivated 24 hours on the slide of room.It is fixed with methanol Cell, and dyed 60 minutes for the rabbit polyclonal antibody of CLD18 with 1 μ g/ml at 25 DEG C.After washing, marked with Alexa488 Goat anti-rabbit igg (Molecular Probes) staining cell, and pass through fluorescence microscopy and evaluated.Fig. 3 display transfections Chinese hamster ovary celI CLD18 is expressed on cell membrane as non-transfected cells.By these heterologous CLD18 expression cells for following It measures, the specificity combined with test antibody.
B. the monoclonal antibody that selection is combined with CLD18/tentatively screened by flow cytometry：
40 hours with encoding human CLD18A2 (SEQ ID NO before the assay：1st, 2) and the expression vector of fluorescent reporter protein Cotransfection HEK293 cells use the HEK293 cells for stablizing expression people CLD18A2 (HEK293-CLD18A2) and use iodine Change the third ingot (PI) to redye.After making cell detachment using 2mM EDTA/PBS, cell is washed, and with complete growth medium with about 1- 5×105Cell is taped against in U-shaped bottom microtiter plate by a cells/well.Cell and doma supernatant are incubated 30 points at 4 DEG C Clock carries out washing step twice, the anti-mouse being finally conjugated with APC or Alexa647 with 1% heat-inactivated FBS/PBS thereafter IgG specific second antibodies are incubated with.Twice after washing step, corotation is fixed with CellFIX (BD Biosciences) The cell of dye.Using BD FACSArray, combined by hybridoma supematant assesse.It is right using the expression of fluorescent marker as horizontal axis The antibody of the longitudinal axis is combined and is mapped.All mouse antibodies 24H5,26B5,26D12,28D10,37G11,37H8,38G5, 38H3,39F11,41C6,42E12,43A11,44E10,47D12,61C2,75B8,85A3,9E8,19B9,45C1,125E1, It is (thin in Fig. 4, Q2 that 163E12,166E2 and 175D10 detect that the surface specific with fluorescent marker expression cell is combined Born of the same parents), for example, containing monoclonal antibody 24H5 (cell in Fig.4A, Q2), 85A3 (Fig.4B), 175D10,125E1, The doma supernatant of 163E12,166E2 and 45C1 (cell in Fig.4C, Q1).
C. compare the combination of the CLD18A2 of antibody and Myc or HA labels：
Further illustrate the binding characteristic of identified CLD18 monoclonal antibody specifics.Therefore, analysis monoclonal resists The combination of body and the CLD18A2 mutant generated by being inserted into epitope tag.CLD18A2-HA(SEQ ID NO：6) exist Containing HA epitope tags in CLD18A2-loop1, and CLD18A2-Myc (SEQ ID NO：4) containing insertion CLD18A2-loop2 In Myc epitope tags.Since the insertion of these labels causes the destruction of epitope, the monoclonal antibody identified can basis It loses and the combination of any mutant is grouped.At 4 DEG C by transient cotransfection fluorescent marker and people CLD18A2, fluorescence mark Note and CLD18A2-HA or the HEK293 cells of fluorescent marker and CLD18A2-Myc containing CLD18 specific monoclonals with resisting The doma supernatant of body is incubated 30 minutes, and the anti-mouse IgG secondary antibodies being conjugated with Alexa647 thereafter are incubated.In BD Before being analyzed on FACSArray, cell is fixed with CellFIX.As exemplified by 24H5,9E8,26B5 and 19B9 in Fig. 5, Can monoclonal antibody be divided by four different groups based on its binding characteristic：(i) with unmodified CLD18A2 and CLD18A2- The antibody that HA and CLD18A2-Myc is combined, such as 24H5, the antibody that (Fig. 5 A) or (ii) are not combined with CLD18A2-HA, such as 9E8, the antibody that (Fig. 5 B) or (iii) are not combined with CLD18A2-Myc, such as 26B5, (Fig. 5 C) or (iv) not with CLD18A2-HA is combined, the antibody also not combined with CLD18A2-Myc, such as 19B9, (Fig. 5 D).
D. compare the combination of antibody and people's CLD18A1 and CLD18A2 transformant by flow cytometry：
Pass through the monoclonal antibody that flow cytometry is identified and the binding specificity of CLD18A2 isoforms.At 4 DEG C By the HEK293 cells for stablizing expression people CLD18A2 (HEK293-CLD18A2) and stablize expression people CLD18A1 (SEQ ID NO： 7th, 8) the HEK293 cells of (HEK293-CLD18A1) with containing monoclonal antibody doma supernatant be incubated 30 minutes, thereafter The anti-mouse IgG secondary antibodies being conjugated with Alexa647 are incubated, and are fixed cell or be not fixed cell and redyed with PI.Make With BD FACSArray, combined by hybridoma supematant assesse.Fig. 6 be shown in 24H5,26B5,26D12,28D10,37G11, 37H8、38G5、38H3、39F11、41C6、42E12、43A11、44E10、47D12、61C2、75B8、85A3、9E8、19B9、 The example of two groups of monoclonal antibodies identified in 45C1,125E1,163E12,166E2,175D10：(i) monoclonal antibody 43A11,45C1 and 163E12 specifically bind people CLD18A2, but do not combine people CLD18A1 (Fig. 6 A, B) and (ii) Dan Ke Grand antibody 37H8 is combined (Fig. 6 A) with two kinds of people's isoforms.
E. antibody is compared by immunofluorescence microscopy and people CLD18A1 and CLD18A2 transfects the combination of body：
With coding CLD18A1 (SEQ ID NO：Or CLD18A2 (SEQ ID NO 8)：2) egg is merged with fluoreporter White expression vector transiently transfects HEK293 cells, and is cultivated on the slide of room.It is not fixed or after paraformaldehyde fixation, 37 DEG C with the tissue culture supernatant staining cell 30 minutes containing monoclonal antibody.It is small with resisting for Alexa555 labels after washing Mouse IgG antibody (Molecular Probes) staining cell.Antibody is assessed by fluorescence microscopy to combine.As shown in fig. 7, antibody 37G11 reacts (Fig. 7 A), but (Fig. 7 B) is not reacted with CLD18A1 with CLD18A2 specificity.On the contrary, antibody 26B5 with CLD18A2 and CLD18A1 reacts (Fig. 8).
For antibody 24H5,26B5,26D12,28D10,37G11,37H8,38G5,38H3,39F11,41C6,42E12, 43A11,44E10,47D12,61C2,75B8,85A3,9E8,19B9 observe that living cells and paraformaldehyde are fixed between cell Apparent dyeing difference.When cell is through fixing, the antibody forms uniform film dyeing (Fig. 7 C, 8C, 8D).It on the contrary, will be living Cell causes the generation of protein cluster with these antibody incubations, it is seen that spot sample staining pattern (Fig. 7 A, 8A, 8B).This display, institute There is antibody to be combined with liver cell surface finding natural epitopes.
F. endogenous expression cell system is determined：
Using CLD18A2 gene-specific primers to (SEQ ID NO in RT-PCR analyses：11,12) it, is expressed with screening The cell line of CLD18A2.It was found that human gastric cancer cell line NCI-SNU-16 (ATCC CRL-5974), NUGC-4 (JCRB0834) and KATO-III (ATCC HTB-103) and human pancreatic cancer cell DAN-G (DSMZ ACC249) shows the strong endogenous expression of CLD18 (Fig. 9).Dyed by using the rabbit polyclonal serum of anti-CLD18 confirms to express in protein level.
G. it dyes endogenous expression cell with CLD18 specific antibodies and carries out immunofluorescence analysis：
DAN-G, SNU-16, NUGC-4 and KATO-III cell are cultivated on the slide of room at the standard conditions.Cell not into Row is fixed or is fixed using methanol, and is dyed with each antibody.To antibody 24H5,26B5,26D12,28D10,37G11,37H8, 38G5,38H3,39F11,41C6,42E12,43A11,44E10,47D12,61C2,75B8,85A3,9E8,19B9 are observed carefully Cellular surface dyes, as shown in Figure 10,11 and 12A.Antibody 45C1,125E1,163E12,166E2 and 175D10 are determined naturally Epitope identifies, and observes that cell surface dyes in unlocked cell, as shown in Figure 12 B.Antibody subgroup, which is shown, mainly to exist Cell-ECM interface or the homologous dyeing of cell membrane in the film free fraction for not adjoining other cells.Other antibody dye Discrete point and aggregation on cell membrane, in a word, it was demonstrated that each antibody is combined with different epitopes, including CLD18 homotypes or abnormal shape Combine covered epitope and easily accessible CLD18 epitopes in established close connection.
H. the cell line of endogenous expression is dyed by flow cytometry：
Pass through the surface table of the CLD18A2 of constitutive expression on flow cytometry KATO-III and NUGC-4 living cells It reaches.For example, dyeing KATO-III and NUGC-4 cells with monoclonal antibody 61C2 or 163E12, it is conjugated with Alexa647 thereafter Anti-mouse IgG secondary antibodies are incubated with, and fix cell or without fixation.It is thin by streaming using BD FACSArray The assessment of born of the same parents' art combines.Figure 13 shows 61C2 and at least 70.3% KATO-III cell combinations, 163E12 and KATO-III and CLD18A2 on NUGC-4 cells is combined.
I. the sequence alignment of mouse and people CLD18A1 and CLD18A2：
People CLD18A2 (NP_001002026) and people CLD18A1 (NP_057453) sequence relatively in N-terminal exist it is poor Different, mouse CLD18 variants (NP_062789 and AAL15636) show intermolecular high homology and sequence variations site (see figure 14)。
J. pass through the reactivity of flow cytometry antibody and mouse CLD18A1 and mouse CLD18A2：
The monoclonal antibody identified by flow cytometry and the combination of mouse CLD18A2 and CLD18A1.By wink When cotransfection fluorescent marker and mouse CLD18A2 (SEQ ID NO：33rd, 35) or fluorescent marker and mouse CLD18A1 (SEQ ID NO：36th, 37) HEK293 cell at 4 DEG C with the monoclonal antibody specifics of CLD18 containing someone respectively 38G5,38H3, The doma supernatant of 37G11,45C1 and 163E12 are incubated 30 minutes, the anti-mouse IgG second being then conjugated with Alexa647 Antibody incubation and fixed cell.Using BD FACSArray, combined by hybridoma supematant assesse.Figure 15 show three kinds it is different Bind profile：38G5 and 45C1 is not combined with any mouse CLD18 isoforms, and 37G11 and 163E12 and mouse CLD18A2 are tied It closes, but is not combined with mouse CLD18A1 and 38H3 is combined with mouse CLD18A1 and CLD18A2.These antibody are in clinic The of great value tool of CLD18 monoclonal antibody genotoxic potentials is determined in preceding research.
In short, these data are shown, monoclonal antibody 24H5,26B5 of the present invention for being generated for CLD18,26D12, 28D10、37G11、37H8、38G5、38H3、39F11、41C6、42E12、43A11、44E10、47D12、61C2、75B8、85A3、 9E8,19B9,45C1,125E1,163E12,166E2 and 175D10 represent more with people CLD18 different epitopes and topology situation Kind binding characteristic.
The combination of different characteristics shown in embodiment 3b, c, d, e, g, h and j can be used for monoclonal antibody being referred to these In inhomogeneity.
4. immunohistochemistry (IHC)
It will be by using SEQ ID NO：21 peptides carry out CLD18A2 epitope-specific antibodies that are immune and generating and are used for The immunohistochemistry characterization of CLD18A2 expression.It is used being sliced from one group of detailed normal and tumor tissues paraffin-embedded tissue In protein expression and positioning analysis.Notable expression is not detected in any other normal organ tissue in addition to stomach (being shown in Table 2, Figure 16 A).On the contrary, expression of the CLD18A2 in various cancers is confirmed by immunohistochemistry, including gastric cancer and lung cancer (Figure 16 B).
It is interesting that expression of the CLD18A2 albumen in gastric mucosa is confined to base portion and depressed area (pit region) stomach In the terminally differentiated cells of epithelium.On the contrary, the cell in gastric mucosa neck region (do carefully by the stomach that entire mucous membrane is particularly supplemented in the area of gorge Born of the same parents) CLD18A2 (Figure 16 C) is not expressed.
Table 2：Pass through expression of the CLD18A2 of IHC analyses in normal and tumor tissues
Monoclonal antibody 39F11 is used for the special Journal of Sex Research of immunohistochemistry CLD18A2.As shown in Figure 17 A, in addition to stomach All tests normal structure in be not detected significant reactive (Figure 17 A), and gastric cancer and lung cancer are still strong positive (figure 17B)。
Another group of antibody of the present invention shows Specific cancer staining pattern, is combined with gastric cancer but reactionless to normal gastric mucosa Property.Monoclonal antibody 26B5 in Figure 18 A show as staining pattern.
Using immunohistochemistry to 175D10 (Figure 18 B), 43A11 on the slice from HEK293 tumor cell lines (Figure 18 C), 163E12 (Fig.18D) and 45C1 (Figure 18 E) carry out specific analysis：Expression people CLD18A2 will be stablized (HEK293-CLD18A2) or CLD18A1 (HEK293-CLD18A1) or expression control plasmid (HEK293- simulations) has been transfected HEK293 tumor cell line heterografts to mouse in, to form solid tumor, contain only and be useful in the expression control plasmid The antibiotics resistance gene of selection.Expression is not detected in the HEK293 xenograft tumours of simulation transfection.On the contrary, It observed strong and uniform film dyeing in HEK293-CLD18A2 xenograft tumours and stomach cancer samples.
5. complement-dependent cytotoxicity (CDC)
A. pass through the CDC of the 1st group of monoclonal antibody of flow cytometry measure：
By from healthy volunteer draw blood to S-Monovette-EDTA vacuum tubes (Sarstedt, Ntirmbrecht, Germany 20 minutes then) are centrifuged to prepare the blood plasma for complement lysis with 600g.Harvest blood plasma is simultaneously stored in -20 DEG C.
In the first set of experiments, it is analyzed doma supernatant for stable expression people CLD18A2 (HEK293- CLD18A2 the ability of HEK293 cell inducing complement dependent cellular cytotoxicities (CDC)).At room temperature by cell with containing respectively The doma supernatant of monoclonal antibody 85A3,28D10,24H5 or 26D12 are incubated 20 minutes.Centrifugation (450g, 5 minutes) removes afterwards Supernatant is removed, in DMEM 20% human plasma (being preheated to 37 DEG C) is added in cell, and is incubated again at 37 DEG C 20 minutes.Its Afterwards, cell cracking is measured with FACS by using propidium iodide (PI) decoration method.Add in PI to 2.5 μ g/ml of final concentration.For stream Formula cell art uses BD FACSArray flow cytometers (BD Biosciences, Mountain View, CA).It collects at least 10000 events are arranged by adjusting preceding lateral scattering (forward sideward scatter, FCS) threshold value for analyzing Except cell fragment.The percentage of lytic cell (PI positive cells) is shown in Figure 19.85A3,28D10 and 26D12 points of monoclonal antibody Not You Dao in HEK293-CLD18A2 cells 33.5%, 38.2% and 39.2% cracking, and the CDC of 24H5 mediations is only 19.3%.
B. the CDC of the 1st group of monoclonal antibody：
In the second set of experiments, specificity of the monoclonal antibody to CLD18A2 expression cells induction CDC is analyzed.Therefore, For a group-specific combination people CLD18A2 or the antibody also in relation with people CLD18A1, it is tested for stable expression people The CDC inductions of the Chinese hamster ovary celI of CLD18A2 (CHO-CLD18A2) or people CLD18A1 (CHO-CLD18A1).24 is small before the assay When, with 3 × 104The density in a/hole drips CHO-CLD18A2 and CHO-CLD18A1 cell inoculations to tissue cultures flat-bottomed microtiter In fixed board.Growth medium is removed within second day, by cell in triplicate with adjusting to containing 10 μ g/ml concentration monoclonal antibodies Doma supernatant is incubated with, the monoclonal antibody be respectively 24H5,26D12,28D10,37G11,37H8,38G5, 38H3,39F11,41C6,42E12,43A11,44E10,47D12 and 61C2.Control cell is with growth medium or containing 0.2% soap The growth medium of angle glycosides is incubated with, and is respectively used to measure background cracking and maximum cracking.It is incubated after twenty minutes, removes at room temperature Supernatant is removed, and in DMEM 20% human plasma (being preheated to 37 DEG C) is added in into cell, is incubated again at 37 DEG C 20 minutes.Then Supernatant is replaced with the PBS containing 2.5 μ g/ml ethidium bromides, measuring fluorescence using Tecan Safire after 520nm excitations sends out It penetrates.Percent specific lysis is calculated as：Specific lytic %=(fluorescent-background fluorescence)/(maximum cracking fluorescence-back of the body Scape fluorescence) × 100.Figure 20 shows that monoclonal antibody 26D12,28D10,37H8,38H3 and 39F11 mediation are for CHO- The height CDC of CLD18A2 cells, monoclonal antibody 38G5 mediate moderate CDC, and monoclonal antibody 41C6 and 61C2 mediation are low CDC, monoclonal antibody 24H5,37G11,42E12,43A11,44E10 and 47D12 do not mediate CDC.On the contrary, all antibody are not The CDC for CHO-CLDA1 cells is can induce, although as by flow cytometry and immunofluorescence assay, 26D12, 28D10,37H8,38H3,39F11,41C6,47D12 and 61C2 are also in relation with CLD18A1.
C. monoclonal antibody titration and the CDC using the 1st group of monoclonal antibody：
In order to measure the ability that anti-CLD18 antibody induces CDC at low concentrations, the reality of three kinds of different antibodies of titration has been carried out It tests.By the CHO-CLD18A2 cells grown in microtiter plate at room temperature with a series of 75B8 of concentration (100,30,10, 3 and 1 μ g/ml), 37H8 (10,3.3 and 1 μ g/ml) and 28D10 (10,1 and 0.1 μ g/ml) be incubated 20 minutes respectively.Remove supernatant In DMEM 20% human plasma (being preheated to 37 DEG C) is added in cell, and be incubated again at 37 DEG C 20 minutes by liquid.It is using Before Tecan Safire are analyzed, supernatant is replaced with the PBS containing 2.5 μ g/ml ethidium bromides.Figure 21 A-C are shown, special Opposite sex cracking percentage is the function of antibody concentration.Monoclonal antibody 75B8 is induced in 10 μ g/ml in CHO-CLD18A2 cells 31.0% cracking, drops to 6.2% (Figure 21 A) in 1 μ g/ml, and monoclonal antibody 28D10 and 37H8 in 1 μ g/ml still The Specific lytic (Figure 21 B, C) of induction 39% and 26.5% respectively.
D. pass through the CDC of the 2nd group of monoclonal antibody of flow cytometry measure：
By from healthy volunteer draw blood to Serum-Monovette vacuum tubes (Sarstedt, N ü rmbrecht, Germany 20 minutes then are centrifuged to prepare the serum for complement lysis with 600g in).Harvest serum is simultaneously stored in -20 DEG C. Control serum heat inactivation 30 minutes at 56 DEG C before preservation.
Analyze KATO-III cell inducing complement dependent cell of the doma supernatant for endogenous expression people CLD18A2 The ability of toxicity (CDC).At 37 DEG C, cell and crude or purifying doma supernatant are incubated 30 minutes, the hybridization Contain monoclonal antibody 45C1,125E1,163E12,166E2 and 175D10 in knurl supernatant respectively.By in RPMI 20% people Serum adds in cell, and is incubated again at 37 DEG C 30 minutes.Thereafter, it using propidium iodide (PI) decoration method, is measured with FACS thin Cellular lysate.Add in PI to 2.5 μ g/ml of final concentration.For flow cytometry, BD FACS Array flow cytometers (BD is used Biosciences、Mountain View、CA).It collects at least 10000 events to be used to analyze, by adjusting preceding lateral scattering (FSC/SSC) threshold value excludes cell fragment.Specific lytic is calculated by following formula：Specific lytic=(the PI in % samples Positive cell-% uses the PI positive cells in the sample of hot inactivated serum).CDC is observed especially for 163E12 to be situated between The fine melt led.
6. antibody-dependent cytotoxicity (ADCC)
It is analyzed doma supernatant for stable expression people CLD18A2 (HEK293-CLD18A2) or people CLD18A1 (HEK293-CLD18A1) ability of HEK293 cell induction of antibodies dependent cellular cytotoxicities (ADCC).
A. it is enriched with human peripheral blood single nucleus cell：
By people's blood from healthy donors, twice, (lymph is thin in Ficoll for haemocyte for dilution in phosphate buffer (PBS) Born of the same parents' isolation medium 1077g/ml, PAA Laboratories, catalog number (Cat.No.) J15-004) higher slice.It is single that blood is detached from interface Nucleus (MNC) washs and is resuspended in the RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-Glutamines and trains It supports in base.
B.ADCC is set：
With Fluorescence Increasing ligand (BADTA, Perkin Elmer cytotoxicity assay kits DELFIA EuTDA Cytotoxicity Reagents, catalog number (Cat.No.) AD0116) mark target cell 30 minutes.With supplemented with 10mM probenecid (Sigma, Catalog number (Cat.No.) P8761), the RPMI-10 of 10-20mMHEPES and 10% heat-inactivated fetal bovine serum is thoroughly after washing, by cell adjust to 1×105A cell/ml.By marked target cell, effector cell (MNC) and adjust to 10 μ g/ml concentration and contain monoclonal The supernatant of antibody is added in round bottom microtiter plate.It is thin using 100: 1 effector cell and target for the effector cell of separation Born of the same parents (E: T) ratio (50: 1 and 25: 1 data are not shown).After being incubated (2 hours, 37 DEG C), stop to measure by centrifuging, and It is discharged in time-resolved fluorescence meter with europium counting to measure the fluorescent ligand in duplex.Cytotoxicity percentage is calculated using following formula Than：Specific lytic %=(experiment discharges counting-spontaneous release and counts)/(maximum release counting-spontaneous release counting) × 100, maximum ligand, which is released through, to be added in Triton X-100 (0.25% final concentration) in target cell and measures, there is no anti- Spontaneous release is measured in the case of body and effector cell.Figure 22 shows, monoclonal antibody 26B5,37H8,38G5,47D12 and 61C2 mediations are for the ADCC of HEK293-CLD18A2 cells.On the contrary, these antibody do not induce notable cell to CLD18A1 targets Toxicity or only induced low levels cytotoxicity, it was demonstrated that the ADCC (Figure 23) of CLD18A2 specificity.
7. Proliferation Ability
Its KATO-III cell growth for inhibiting endogenous expression people CLD18A2 is analyzed the mouse monoclonal antibody of purifying Ability.
1 × 10 is cultivated in the presence of about 10 μ g monoclonal antibodies4Target cell (the KATO- of a endogenous expression CLD18A2 III)。
DELFIA cell proliferation reagents box (Perkin-Elmer, catalog number (Cat.No.) AD0200) is to be based on measuring being proliferated in microwell plate The heterotope immunoassays that the bromo- 2 '-BrdUs of 5- (BrdU) mix in cell DNA building-up process.The BrdU of incorporation is used The monoclonal antibody of europium label is detected.To carry out antibody test, cell and denatured DNA are fixed using fixer.It washes away not Binding antibody, and add DELFIA inducers europium is dissociated in solution from labelled antibody, they in the solution with DELFIA The component of inducer forms high fluorescent chelate.Measured fluorescence (usage time resolved fluorometric art in the detection) and each hole The DNA synthesis of middle cell is proportional.
Strong proliferation suppression observed respectively for antibody 125E1,163E12,45C1,37G11,37H8,28D10 and 166E2 System.Moderate Proliferation Ability observed respectively for mouse antibodies 43A11,175D10,42E12,26D12,61C2 and 38H3.
8. the performance in therapeutic mice xenograft model
Controlling for the monoclonal antibody of identified specific binding CLD18A2 is had studied in therapeutic heteroplastic transplantation model Treat potentiality.
A. the high early treatment for expressing CLD18A2 tumours in mouse
With 1 × 107The HEK293 cell subcutaneous inoculations of a stable expression high level people CLD18A2 (HEK293-CLD18A2) SCID mice.Expressions of the people CLD18A2 in HEK293-CLD18A2 cells in the primary gastric cancer from patient Expression it is suitable.Each experimental therapy group includes 10 mouse (the number of mice n=10 in every group).Exist to the treatment of mouse Start within 3 days after tumor inoculation.In 4 weeks, it is injected intravenously the doma supernatant of 200 μ g purifying once a week, represents small Mouse monoclonal antibody 26B5,26D12,28D10,37G11,37H8,38G5,39F11,42E12,43A11,38H3 or 61C2.Or Person was applied twice weekly by alternate injection in intravenous and peritonaeum containing mouse monoclonal antibody 45C1 in 6 weeks, The doma supernatant of the 200 μ g purifying of 125E1,163E12,166E2 or 175D10.Monitoring treated mice is swollen twice a week Knurl grows (gross tumor volume=length x width x width/2, with mm3Meter).If gross tumor volume reaches 500mm3Or it is falling ill Under serious situation, then mouse is put to death.Figure 24 illustrates antibody strong inhibition HEK293-CLD18A2 tumour cells of the present invention Growth.Figure 25 A and 25B are shown, in the early treatment heteroplastic transplantation model of application HEK293-CLD18A2, by using the present invention Antibody carries out treatment and extends survival.
B. the tardy treatment of the high CLD18A2 expression tumour of mouse middle and advanced stage
Design similarly based on the Tumor Xenograft Models of HEK293-CLD18A2 as tardy therapeutic scheme rather than Above-mentioned early treatment.Mouse is grouped at random in the test group respectively containing 5-6 mouse, and make within the 27th day after tumor inoculation The doma supernatant initial treatment purified with 200 μ g, the doma supernatant contain mouse monoclonal antibody respectively 43A11,163E12 and 175D10.In six weeks, carry out administration of antibodies twice weekly by alternate injection in intravenous and peritonaeum. In this model, antibody of the invention also shows inhibition tumour growth.For some antibody, which results in the extension (figures of survival 26)。
C. the early treatment of the tumour of expression low-level CLD18A2
2 × 10 are inoculated to SCID mice5A DAN-G tumor cell lines, the cell line are constitutive expression low-levels The wellability human pancreatic cancer cell of CLD18A2 albumen.The treatment (10/group) of 3 days founders after tumour transplatation：Six In week the doma supernatant of 200 μ g purifying, the hybridization are applied twice weekly by alternate injection in intravenous and peritonaeum Knurl supernatant contains mouse monoclonal antibody 45C1,125E1,163E12,166E2 or 175D10.Since pancreas DAN-G tumours are thin Born of the same parents tie up to internal aggressiveness and rapid tumour growth, and tumor cachexia occurs for mouse, and dead in several days.Although because The window that this measures curative effect is very narrow, but also observed in this model the antibody-mediated Tumor growth inhibition of the present invention with And prolonging survival (Figure 27 A and 27B).
D. antibody of the invention does not cause side effect in mouse
Using mouse CLD18A2 specific primers to (justice in RT-PCR analyses：CTA CCA AGG GCT ATG GCG TTC, antisense：GCA CCG AAG GTG TAC CTG GTC) from one group of extensive normal mouse tissues expand cDNA (see Figure 28).
Other than stomach, mouse CLD18A2 expression is can't detect in any normal structure tested (see Figure 28).This Outside, it is carried out in a variety of normal mouse tissues using with the CLD18A2 specific antibodies of people and mouse CLD18A2 cross reactions The immunohistochemical analysis (being shown in Table 3) of CLD18A2 expression.Other than normal gastric mucosa, the normal structure of all tests is equal Do not show that CLD18A2 is expressed.As we are to as observed by people CLD18A2, we are for mouse counterpart, it was also found that the greatest extent Pipe surface epithelium and the pit cell of deeper inside express CLD18A2, but the neck in center is cloudy for CLD18A2 in its cell surface Property (see Figure 29 A-C).In short, the Tissue distribution of CLD18A2 is apparently identical in people and mouse.
Table 3：Pass through expression of the immunohistochemical analysis CLD18 in mouse normal structure
We have also investigated the potential side effects that antibody 125E1,163E12,166E2 and 175D10 are mediated in mouse.Institute There is the previously passed facs analysis of these antibody to show to react with mouse CLD18A2 and people's albumen.
Any visible side effect is not observed using during these Antybody therapies and after treatment, it is small with Antybody therapy Any and relevant tectology feelings of toxicity are also not observed compared with not treating (PBS processing) mouse in mouse in gastric mucosa Condition (see Figure 30).
9. antibody is chimeric
A. mouse/people's chimeric mAb is generated
By standard method well known by persons skilled in the art, from human peripheral blood single nucleus cell (PBMC) and people's spleen tissue In prepare total serum IgE, prepare single-stranded cDNA thereafter, such as use RNeasy small volume of reagent box (Qiagen) and Superscript II Reverse transcriptase (Invitrogen).
The constant region of human kappa light chain is expanded from PBMC cDNA by PCR.Sense oligomer (SEQ ID NO：38) in perseverance 5 ' the ends for determining area add in BamHI restriction sites, and by the original nucleic acid of encoding constant regions the first two amino acid (Arg-Thr) Sequence 5 '-CGAACT-3 ' becomes 5 '-CGTACG-3 ', generates BsiWI restriction sites without changing amino acid sequence.Antisense is few Polymers (SEQ ID NO：39) comprising terminator codon, and NotI restriction sites are added in 3 ' ends of expanded constant region. By PCR product and standard expression vectors (such as pcDNA3.1 (+), Invitrogen) successively with BamHI and NotI restriction enzymes one It rises and is incubated.In addition carrier is handled with calf intestinal alkaline phosphatase, to prevent recirculation.Finally constant region is connected into carrier In, so as to variable region thereafter in any fusion before constant region now possibly through in remaining vector multiple cloning site HindIII restriction sites (5 '-AAGCTT-3 ') and PCR product in the BsiWI restriction sites (5 '-CGTACG- that generate 3 ') it carries out.It is inserted into the sequence such as SEQ ID NO of the human kappa light chain constant region in the carrier：Listed by 40, the ammonia of the people's κ constant regions Base acid sequence such as SEQ ID NO：Listed by 41.
The constant region of people's γ -1 heavy chains is expanded from spleen cDNA by PCR.Sense oligomer (the SEQ ID of 5 ' phosphorylations NO：42) it is placed on the naturally occurring ApaI restriction sites of 11, constant region starting point downstream nucleotide, and is being expanded Constant region 5 ' ends add in HindIII restriction sites.Antisense scant polymer (the SEQ ID NO of 5 ' phosphorylations：43) NotI restriction sites are added in comprising terminator codon, and in the 3 ' of the constant region ends expanded in this way.Make the PCR generated in this way Product becomes flat end, and 5 ' phosphorylations.By using distinction antisense scant polymer (SEQ ID NO：44) PCR and pass through Sequencing confirms that the γ constant regions of amplification are IgG1 subclass.It will carry different from antibiotic resistance in light chain expression used carrier (such as Neomycin) antibiotic resistance (such as hygromycin) standard expression vectors (such as pcDNA3.1 (+)/Hygro, Invitrogen) It is incubated with PmeI restriction enzymes, to remove multiple cloning sites completely, leaves flat end.In addition it is handled with calf intestinal alkaline phosphatase Carrier, to prevent recirculation.Finally constant region is connected into carrier, so as to which variable region is any before constant region thereafter The HindIII restriction sites (5 '-AAGCTT-3 ') and limited by ApaI that fusion generates now possibly through PCR product Property site (5 '-GGGCCC-3 ') processed is realized.Confirm that constant region is correctly orientated in the carrier by being sequenced, that is, being suitable for should The orientation of above-mentioned promoter in carrier.Due to the position of ApaI restriction sites, any variable region amplification for this purpose is equal Preceding 11 nucleotide in people's γ -1 constant-region sequences in addition to ApaI sites need to be included.The people expanded in this way being inserted into carrier γ -1 heavy chain constant region sequences such as SEQ ID NO：Listed by 45, the amino acid sequence such as SEQ for people's γ -1 constant regions expressed in this way ID NO：Listed by 46.
Table 4：For the mouse hybridoma cell system of antibody cloning
Corresponding to its mouse counterpart, chimeric mAb is named by prefixing " ch- ", such as ch-43A11, ch-163E12、ch-125E1、ch-166E2、ch-175D10、ch-45C1。
The amplification of mouse light chain and heavy chain variable region according to Matz etc. (Nucleic Acids Research, the 1999, the 27th Volume, No.6) description " step-out PCR " method carry out.For this purpose, by standard method well known by persons skilled in the art from list Total serum IgE is prepared in cloned hybridoma cell system (being shown in Table 4), such as uses RNeasy small volume of reagent box (Qiagen).According to " mould Plate-conversion " method prepares single-stranded cDNA, also by Matz etc. (Nucleic Acids Research, volume 1999,27, No.6, 1558) it describes.In addition to (dT) 30 oligomer (SEQ ID NO：47) other than, it further includes DNA/RNA hybridization oligomers (SEQ ID NO：48) as 5 ' connectors, for the template switch in the polymerization process of cDNA chains.In this connector oligomer, last 3 A nucleotide replaces deoxyribonucleotide with ribonucleotide.Thereafter " step-out PCR " is permanent using targeting mouse κ chains Determine area or targeting γ chains 1,2a or 3 subclass (are respectively SEQ ID NO：49 to 52) antisense scant polymer of constant region.By miscellaneous The IgG subclass mouse monoclonal antibody that oncocyte system generates is handed over previously to carry out immune analysis (see embodiment with IsoStrip 1) appropriate antisense scant polymer (being shown in Table 4), is correspondingly selected.Primer mixture is in " step-out PCR " as just oligomerization Object, it includes SEQ ID NO：Two kinds of oligomers listed by 53 and 54.Some hybridoma cell lines express more than one heavy chain Or light chain (other than the chain for being used to generate expressed by the myeloma cell line of hybridoma).What table 4 summarized clone and was sequenced Mouse antibodies chain variable region (SEQ ID NO：55 to 69 and SEQ ID NO：132 to 146) it is fitted into the overall length cloning and be sequenced Antibody chain (SEQ ID NO：100to 129) SEQ ID NO.
Then by omitting 5 ' UTR and 3 ' murine constant regions and being reflected in the PCR of end addition restriction site to expand Fixed mouse variable region, the restriction site allow to be subcloned into the expression vector of prepared human constant regions.This Outside, sense oligomer provides shared Kozak sequences (5 '-GCCGCCACC-3 ' or 5 '-AGCCACC-3 '), for weight chain variable The antisense scant polymer in area (is shown in Table 4, SEQ ID comprising 11 nucleotide before people's γ -1 constant regions in addition to ApaI restriction sites NO：70 to 99).Using HindIII and BsiWI restriction enzymes clone's kappa light chain variable area, gamma heavy chain variable region needs HindIII With ApaI restriction enzymes.Contain internal HindIII restriction sites in the gamma heavy chain variable region of monoclonal antibody 45C1 --- this When replaced using compatible BsaI enzymes (see SEQ ID NO：80).SEQ ID NO：100 to 114 display gained chimeric antibodies Nucleic acid sequence (is shown in Table 4).SEQ ID NO：The amino acid sequence (being shown in Table 4) of chimeric antibody that 115 to 129 displays are accordingly expressed.
B. it generates and produces the chimeric antibody for CLD18
Produce the mammal cell line of chimeric antibody of the production with CLD18 specificity.The cell line derives from HEK293T cells (ATCC CRL-11268).The day before transfection, by 2.5 × 107A cell is layered in 14.5cm tissue culture dishes And it is cultivated in 20ml complete mediums or by 1 × 107A cell is layered in 10cm tissue culture dishes and is trained completely in 10ml It supports and is cultivated in base or by 0.6 × 106A cell is layered in 12 hole tissue culturing plates and is cultivated in 2-3ml complete mediums (complete medium：Antibiotic-free DMEM supplemented with 10%FBS:F12 culture mediums).The cell density recommended during transfection should be 90% converges.Culture medium is replaced with into fresh culture before transfection is faced.HEK293T cells are transfected with transfection reagent, such as Lipofectamine 2000 (Invitrogen, 11668-019) or polyethyleneimine (PEI) (Sigma-Aldrich, 408727).The example of the transfection of HEK293T cells is that the total DNA measured as 110 μ g or 296 μ g is used for 14.5cm culture dishes, The ratio of transfection agents and DNA are respectively 1: 2.5 and 1: 12 for Lipofectamine 2000 and PEI.It 24 hours after transfection will Culture medium replaces with GMP and is applicable in culture medium, such as the culture medium that X-Vivo 15 (Cambrex) or chemical composition determine, such as nothing The Pro293a (Cambrex) of serum.The transfection HEK293T cells generated for the chimeric mAb of CLD18 are further cultured for 96 hours.Crude supernatant is harvested, be sterile filtered and is purified by Protein A-agarose.It is dense that antibody is measured by BCA measure Degree, passes through sodium dodecyl sulfate gel electrophoresis and coomassie chromoscopy purity.
C. the binding characteristic of chimeric mAb
As described in Example 3, that clones and generate by flow cytometry resists for CLD18A2 chimeric monoclonals Body.By the HEK293 living cells for stablizing expression people CLD18A2 (HEK293-CLD18A2) and stable expression people at 4 DEG C CLD18A1(SEQ ID NO：7th, 8) the HEK293 cells of (HEK293-CLD18A1) with containing chimeric mAb through pure The HEK293T cell culture supernatants of change are incubated 30 minutes, the F (ab ') being conjugated with APC thereafter2Segment Goat anti-Human IgG Fc γ secondary antibodies are incubated, and are redyed with PI.Using BD FACSArray, combined by hybridoma supematant assesse.
Similarly, by flow cytometry it is endogenous expression CLD18A2 human tumor cell line, such as KATO-III and NUGC-4 cells.
Figure 31 A and B show chimeric antibody ch-43A11, ch-125E1, ch-163E12, ch-166E2 and ch-175D10's Flow cytometry.They show that natural epitopes identify, and show to CLD18A2 rather than CLD18A1 expression cells Strong and specific combination.
D. complement-dependent cytotoxicity (CDC)
By from healthy volunteer draw blood to Serum-Monovette vacuum tubes (Sarstedt, N ü rmbrecht, Germany 20 minutes then are centrifuged to prepare the serum for complement lysis with 600g in).Harvest serum is simultaneously stored in -20 DEG C. Control serum heat inactivation 30 minutes at 56 DEG C before preservation.
Its KATO- for endogenous expression people CLD18A2 is analyzed the chimeric antibody of the present invention of Protein A-agarose purifying The ability of the CHO-CLD18A2 cell inducing complement dependent cellular cytotoxicities (CDC) of III cells and stable transfection.At 37 DEG C Lower ch-163E12, ch-166E2 and ch-175D10 points of the monoclonal antibody by cell and 2.5 μ g/ml of final concentration to 35 μ g/ml It Fu Yu not be 30 minutes.20% human serum in RPMI is added in cell, is incubated again at 37 DEG C 30 minutes.Thereafter, pass through end The PI of a concentration of 2.5 μ g/ml dyes to distinguish dead cell and living cells, is split by the antibody-mediated cell of Flow Cytometry Assay Solve percentage.For flow cytometry, BD FACSArray flow cytometers (BD Biosciences, Mountain are used View, CA).At least 10000 events are collected for analyzing, it is thin to exclude by adjusting preceding lateral scattering (FSC/SSC) threshold value Born of the same parents' fragment.Specific lytic is calculated by following formula：Specific lytic=(the PI positive cells-% in % samples is inactivated using heat PI positive cells in the sample of serum).Several antibody are shown with the Specific lytic of CDC mediations.All three antibody is equal The cell-mediated strong CDC (Figure 32) to CHO-CLD18A2.For KATO-III cells, antibody ch-163E12 and ch-175D10 are The inducer of strong CDC.
E. antibody-dependent cytotoxicity (ADCC)
Its KATO-III cell for endogenous expression people CLD18A2 is analyzed the chimeric antibody of the present invention of FPLC purifying to lure Lead the ability of antibody-dependent cytotoxicity (ADCC).
By people's blood from healthy donors, twice, haemocyte is in Ficoll (1077g/ for dilution in phosphate buffer (PBS) Ml, Pharmacia) higher slice.After centrifugation, separating peripheral blood mononuclear cells (PBMC) from interface is washed and is resuspended in benefit It has filled in the X-Vivo-15 culture mediums of 5% heat-inactivated fetal bovine serum.
15 hours before measure, with luciferase transfection KATO-III cells, and with 5 × 104It is micro- that a cells/well is layered on white In orifice plate.
For this measure, effector cell (PBMC, such as above-mentioned system are added in effector cell and target cell (E: T) ratio 20: 1 It is standby) and FPLC purifying chimeric antibody, and in 37 DEG C, 5%CO2It is lower to be incubated 2-3 hours.Final concentration of 50 μ of the antibody in hole g/ml.After preincubate 2-3 hours, fluorescein (BD Biosciences, San Jose USA) is added in 1mg/ml.Use micropore Plate reader (Infinite200, Tecan, Switzerland) is continuously measured produced by living cells luciferase oxyluciferin Huang Luminous up to 6 hours.Percentage of cytotoxicity is calculated using following formula：Specific lytic %=100- ((sample shine counting- Spontaneous luminescence counts)/(the luminous counting-spontaneous luminescence of maximum counts) × 100), spontaneous luminescence is by adding in Triton X-100 (0.2% final concentration) measures, peak signal is measured in the case of there is no antibody.
Use this measure display, monoclonal antibody ch-163E12 and ch-175D10 cell-mediated strong ADCC to KATO-III (Figure 33).
F. Proliferation Ability
The thin of its KATO-III cell for inhibiting endogenous expression CLD18A2 is analyzed the chimeric antibody of the present invention of FPLC purifying The ability of intracellular growth.It is (real see the Proliferation Ability of mouse antibodies that target cell (KATO-III) is cultivated in the presence of each chimeric antibody Apply example 7).Show that the chimeric antibody ch-163E12 and ch-166E2 of FPLC purifying inhibit cell Proliferation.
10. antibody is selected as candidate clinical pilot drug
Ideal clinical pilot drug can cover a variety of treatments and diagnostic application (referring also to the purposes of IV chapters and sections-present invention And method).According to the present invention, the antibody for CLD18-A2 is provided.It has been shown that according to antibody provided by the invention in spy The opposite sex provides wide spectrum in specific tumors cell in terms of the cell Proliferation of induction CDC and ADCC and inhibition expression CLD18 Characteristic.Also, it has proven that the chimeric of antibody can be allowed to obtain the other Fc dependences effect being not present in parent mouse molecule Answer function.For example, herein it has been shown that the antibody 175 D10 with mouse IgG 1 not inducing complement dependent cellular cytotoxicity (see Embodiment 5), and the tumour cell of the cracking constitutive expression of the ch-175D10 inducing specifics with human IgG1 CLD18 (is shown in Table 5 With table 6).
Antibody provided by the invention can be according to the ability of its binding characteristic and the cell-mediated effector function to expressing CLD18 And it is referred in different classes.It can be based on its functional character and candidate clinical pilot medicine is selected in antibody provided by the invention Object.The general introduction of the characteristic of selected mouse of the present invention and chimeric antibody provides in table 5 and table 6 respectively.
The candidate clinical pilot drug of the present invention can have one or more following characteristics：
A) with reference to people CLD18A2, but do not combine people CLD18A1 (such as 43A11,45C1,125E1,163E12,166E2 and 175D10 and ch-43A11, ch-45C1, ch-125E1, ch-163E12, ch-166E2 and ch-175D10).Such as see figure 6A and 6B.
B) with reference to mouse CLD18A2, but do not combine mouse CLD18A1 (such as 125E1,163E12,166E2 and 175D10).Such as see Figure 15 A and 15B.
C) naturally express with reference to tumour cell CLD18 (such as 45C1,43A11,125E1,163E12,166E2 and 175D10 and ch-45Cl, ch-43A11, ch-125E1, ch-163E12, ch-166E2 and ch-175D10).Such as see figure 13。
D) with reference to the CLD18 (such as 45C1,43A11,125E1,163E12,166E2 and 175D10) in intercellular contacts area. Such as see Figure 12 A and 12B.
E) mediation CDC induction killing expression CLD18 cell (such as 45C1,125E1,163E12,166E2 and 175D10 and ch-163E12 and ch-175D10).Such as see Figure 32.
F) cell (such as ch-163E12 and ch-175D10) of the killing expression CLD18 of mediation ADCC inductions.Such as see Figure 33.
G) inhibit cell Proliferation (such as 45C1,125E1,163E12,166E2 and 175D10 and the ch- of expression CLD18 163E12 and ch-166E2).
H) use expression CLD18 cell heteroplastic transplantation model in inhibit tumour growth (such as 43A11,125E1, 163E12,166E2 and 175D10).Such as see Figure 24.
I) use expression CLD18 cell heteroplastic transplantation model in extend survival (such as 43A11,125E1, 163E12,166E2 and 175D10).Such as see Figure 25 B.
For the characteristic of candidate lead drug to be selected to summarize
Table 5：Mouse antibodies
Legend：+ excellent performance, the performance of (+) in different condition.
Table 6：Chimeric antibody
Legend：+ excellent performance, the performance of (+) in different condition, n.d. are not carried out
New international patent application
Ganymed Pharmaceuticals AG etc.
" monoclonal antibody for claudin -18 for being used for treating cancer "
Our reel number：342-31 CNT2
Biomaterial additional page
The proof of other preserved materials：
1) preserved material (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC-2745, DSM ACC2746, DSM ACC2747, DSM ACC2748) preservation mechanism title and Address is：
Germany Microbiological Culture Collection Center
Mascheroder Weg 1b
38124Braunschweig
DE
2) title of preserved material (DSM ACC2808, DSM ACC2809, DSM ACC2810) preservation mechanism and address are：
Germany Microbiological Culture Collection Center
Inhoffenstr.7 B
38124 Braunschweig
DE
To all other explanations for being previously mentioned preserved material
Mouse (Mus musculus) the myeloma P3X63 Ag8U.1 merged with mouse (Mus musculus) splenocyte
The hybridoma of the anti-human claudin -18A2 antibody of secretion
3) preservation people：All above-mentioned preservations are carried out by following preservation people：
Ganymed Pharmaceuticals AG
Freiligrathstrafle 12
55131 Mainz
DE
Applicant or procuratorial document number 342-31PCT International application no PCT/EP2006/011302
It is related to the explanation of conserving microorganism or other biological material
(the 13rd article of PCT)
PCT/RO/134 tables (in July, 1998；In January, 2004 reprints)
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The budapest treaty of the microbial preservation international recognition of proprietary program
International table
Survival proves
It is indicated by nextpage bottom
Ganymed Pharmaceuticals AG's International Depository Authority
Freiligrathstr.12 is issued according to the 2nd section of the 10th article
55131 MainZ
1Represent the first submission date or if new submit or shift, the last corresponding date is (new to submit or turn Move the date)
2Suitable for clause 10.2.a) ii) and iii), mark nearest viability test
3Cross mark application type
DSMZ-BP/4 tables (homepage) 12/2001
The budapest treaty of the microbial preservation international recognition of proprietary program
International table
Ganymed Pharmaceuticals AG
Freiligrathstr.12
55131 MainZ
1If clause 6.4.d) it is applicable in, which has as obtained the date of International Depository Authority state.
DSMZ-BP/4 tables (single page) 12/2001
The budapest treaty of the microbial preservation international recognition of proprietary program
International table
Survival proves
It is indicated by nextpage bottom
Ganymed Pharmaceuticals AG's International Depository Authority
Freiligrathstr.12 is issued according to the 2nd section of the 10th article
55131 MainZ
1Represent the first submission date or if new submit or shift, the last corresponding date is (new to submit or turn Move the date)
2Suitable for clause 10.2.a) ii) and iii), mark nearest viability test
3Cross mark application type
DSMZ-BP/4 tables (homepage) 12/2001
The budapest treaty of the microbial preservation international recognition of proprietary program
International table
Ganymed Pharmaceuticals AG
Freiligrathstr.12
55131 MainZ
1If clause 6.4.d) it is applicable in, which has as obtained the date of International Depository Authority state.
DSMZ-BP/4 tables (single page) 12/2001
The budapest treaty of the microbial preservation international recognition of proprietary program
International table
Survival proves
It is indicated by nextpage bottom
Ganymed Pharmaceuticals AG's International Depository Authority
Freiligrathstr.12 is issued according to the 2nd section of the 10th article
55131 MainZ
1Represent the first submission date or if new submit or shift, the last corresponding date is (new to submit or turn Move the date)
2Suitable for clause 10.2.a) ii) and iii), mark nearest viability test
3Cross mark application type
DSMZ-BP/4 tables (homepage) 12/2001

Claims (9)

1. it combines CLD18A2 and the chimeric antibody of the cell of killing expression CLD18A2 is mediated to prepare for preventing or treat cancer Purposes in the drug of disease,
Wherein described chimeric antibody is such albumen, it includes the combination of heavy chain variable region (VH) and light chain variable region (VL), The combination respectively contains CDR1, CDR2 and CDR3 complementarity-determining region group selected from following (i) to (ix)：
(i)VH：CDR1：SEQ ID NO：115 45-52 positions, CDR2：SEQ ID NO：115 70-77 positions, CDR3：SEQ ID NO：115 116-125 positions, VL：CDR1：SEQ ID NO：122 49-53 positions, CDR2：SEQ ID NO：122 71-73 positions, CDR3：SEQ ID NO：122 110-118 positions,
(ii)VH：CDR1：SEQ ID NO：116 45-52 positions, CDR2：SEQ ID NO：116 70-77 positions, CDR3：SEQ ID NO：116 116-126 positions, VL：CDR1：SEQ ID NO：121 47-58 positions, CDR2：SEQ ID NO：121 76-78 Position, CDR3：SEQ ID NO：121 115-123 positions,
(iii)VH：CDR1：SEQ ID NO：117 45-52 positions, CDR2：SEQ ID NO：117 70-77 positions, CDR3：SEQ ID NO：117 116-124 positions, VL：CDR1：SEQ ID NO：123 47-52 positions, CDR2：SEQ ID NO：123 70-72 Position, CDR3：SEQ ID NO：123 109-117 positions,
(iv)VH：CDR1：SEQ ID NO：119 44-51 positions, CDR2：SEQ ID NO：119 69-76 positions, CDR3：SEQ ID NO：119 115-125 positions, VL：CDR1：SEQ ID NO：126 47-58 positions, CDR2：SEQ ID NO：126 76-78 Position, CDR3：SEQ ID NO：126 115-122 positions,
(v)VH：CDR1：SEQ ID NO：118 45-52 positions, CDR2：SEQ ID NO：118 70-77 positions, CDR3：SEQ ID NO：118 116-126 positions, VL：CDR1：SEQ ID NO：125 47-58 positions, CDR2：SEQ ID NO：125 76-78 positions, CDR3：SEQ ID NO：125 115-123 positions,
(vi)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：124 47-58 positions, CDR2：SEQ ID NO：124 76-78 Position, CDR3：SEQ ID NO：124 115-123 positions,
(vii)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：127 47-58 positions, CDR2：SEQ ID NO：127 76-78 Position, CDR3：SEQ ID NO：127 115-123 positions,
(viii)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：128 47-58 positions, CDR2：SEQ ID NO：128 76-78 Position, CDR3：SEQ ID NO：128 115-123 positions and
(ix)VH：CDR1：SEQ ID NO：120 45-53 positions, CDR2：SEQ ID NO：120 71-78 positions, CDR3：SEQ ID NO：120 117-128 positions, VL：CDR1：SEQ ID NO：129 47-52 positions, CDR2：SEQ ID NO：129 70-72 Position, CDR3：SEQ ID NO：129 109-117 positions.
2. the purposes of claim 1, wherein the cancer be selected from gastric cancer, the cancer of the esophagus, cancer of pancreas, lung cancer, oophoroma, colon cancer, Liver cancer, head and neck cancer and gallbladder cancer.
3. the purposes of claims 1 or 2, wherein the cancer is gastric cancer or cancer of pancreas.
4. the purposes of any one of claims 1 to 3, wherein the chimeric antibody is single chain protein, wherein VL the and VH areas Pairing forms monovalent molecules.
5. the purposes of any one of claims 1 to 4 wherein the antibody includes heavy chain variable region (VH), contains selected from SEQ ID NO：132,133,134,135,136 and 137 amino acid sequence.
6. the purposes of any one of claim 1 to 5 wherein the antibody includes light chain variable region (VL), contains selected from SEQ ID NO：138,139,140,141,142,143,144,145 and 146 amino acid sequence.
7. the purposes of any one of claim 1 to 6, wherein the antibody includes the weight to (ix) selected from following possibility (i) Chain variable region (VH) and the combination of light chain variable region (VL)：
(i) comprising SEQ ID NO：The VH of amino acid sequence shown in 132 and include SEQ ID NO：Amino acid sequence shown in 139 VL,
(ii) comprising SEQ ID NO：The VH of amino acid sequence shown in 133 and include SEQ ID NO：Amino acid sequence shown in 138 The VL of row,
(iii) comprising SEQ ID NO：The VH of amino acid sequence shown in 134 and include SEQ ID NO：Amino acid sequence shown in 140 The VL of row,
(iv) comprising SEQ ID NO：The VH of amino acid sequence shown in 136 and include SEQ ID NO：Amino acid sequence shown in 143 The VL of row,
(v) comprising SEQ ID NO：The VH of amino acid sequence shown in 135 and include SEQ ID NO：Amino acid sequence shown in 142 VL,
(vi) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 and include SEQ ID NO：Amino acid sequence shown in 141 The VL of row,
(vii) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 and include SEQ ID NO：Amino acid sequence shown in 144 The VL of row,
(viii) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 and include SEQ ID NO：Amino acid shown in 145 The VL of sequence and
(ix) comprising SEQ ID NO：The VH of amino acid sequence shown in 137 and include SEQ ID NO：Amino acid sequence shown in 146 The VL of row.
8. the purposes of any one of claim 1 to 7, wherein the antibody includes the weight to (ix) selected from following possibility (i) The combination of chain and light chain：
(i) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 115 and include SEQ ID NO：Amino acid sequence shown in 122 The light chain of row,
(ii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 116 and include SEQ ID NO：Amino acid shown in 121 The light chain of sequence,
(iii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 117 and include SEQ ID NO：Amino acid shown in 123 The light chain of sequence,
(iv) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 119 and include SEQ ID NO：Amino acid shown in 126 The light chain of sequence,
(v) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 118 and include SEQ ID NO：Amino acid sequence shown in 125 The light chain of row,
(vi) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 124 The light chain of sequence,
(vii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 127 The light chain of sequence,
(viii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino shown in 128 The light chain of acid sequence and
(ix) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 129 The light chain of sequence.
9. include the antibody selected from following possibility (i) to the combination of the heavy chain and light chain of (ix)：
(i) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 115 and include SEQ ID NO：Amino acid sequence shown in 122 The light chain of row,
(ii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 116 and include SEQ ID NO：Amino acid shown in 121 The light chain of sequence,
(iii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 117 and include SEQ ID NO：Amino acid shown in 123 The light chain of sequence,
(iv) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 119 and include SEQ ID NO：Amino acid shown in 126 The light chain of sequence,
(v) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 118 and include SEQ ID NO：Amino acid sequence shown in 125 The light chain of row,
(vi) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 124 The light chain of sequence,
(vii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 127 The light chain of sequence,
(viii) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino shown in 128 The light chain of acid sequence and
(ix) comprising SEQ ID NO：The heavy chain of amino acid sequence shown in 120 and include SEQ ID NO：Amino acid shown in 129 The light chain of sequence.
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